# Old Russian Translation of *Cosmographia, sive De Situ Orbis* by Pomponius Mela: Reception of Renaissance Culture in Muscovy (15th-17th Centuries)

Tatiana Matasova

## 1. Introduction and main purpose of the paper

For a long time the question of contacts between Muscovy and Western Europe during the reign of Ivan III (1462-1505) and his successors has attracted attention of different researchers. It has been noted that during the last quarter of the 15th to the first third of the 16th century Muscovite culture was developing under a strong Renaissance influence. A variety of elements of Renaissance culture were assimilated by Russia and found vivid reflection in Russian architecture, literature, and fine arts. Renaissance traces are evident in the exterior of the architectural ensemble of the Kremlin. One of the most important questions related to the Renaissance influence on Russian culture is the issue of the degree and nature of these changes: whether they were integral and fundamental, affecting the sole basics of Muscovite culture, or whether they only touched the surface of Russian way of life, not altering traditions and ideas1 . In order to solve this matter one might find it helpful to turn to the history of the

1 Chreptovič-Butenev 1909; Beltrami 1912; Filippini 1925; Lo Gatto 1934; Shmurlo 1937; Barbieri 1957; Gukowsky 1967; Sinicyna 1977; Choroškevič 1980; Baracchi 1983; Zemcov, Glazyčev 1985; Pod"japol'skij 1986; Chreptovič-Butenev 1993; Sinicyna 1993; Mil'čik 1997; Panova 1998; Pod"japol'skij 2000; Višnevskaja 2004; Mel'nikova 2006;

Tatiana Matasova, Lomonosov Moscow State University, Russian Federation<EMAIL_ADDRESS>0000-0002-9433-5068

FUP Best Practice in Scholarly Publishing (DOI 10.36253/fup\_best\_practice)

Tatiana Matasova, *Old Russian Translation of* Cosmographia, sive De Situ Orbis *by Pomponius Mela: Reception of Renaissance Culture in Muscovy (15th-17th Centuries)*, pp. 37-52, © 2020 Author(s), CC BY 4.0 International, DOI 10.36253/978-88-5518-198-3.03, in Giovanna Siedina (edited by), *Essays on the Spread of Humanistic and Renaissance Literary Civilization in the Slavic World (15th-17th Century)*, © 2020 Author(s), content CC BY 4.0 International, metadata CC0 1.0 Universal, published by Firenze University Press (www.fupress.com), ISSN 2612-7679 (online), ISBN 978-88-5518-198-3 (PDF), DOI 10.36253/978-88-5518-198-3

most significant Renaissance texts – the ways in which they appeared in Moscow, who and how made use of them, and how these texts were perceived by Muscovite intellectuals.

The studies on the distribution of Renaissance texts through the territory of the Grand Duchy of Moscow are at the center of attention in Russia during the last years. This especially relates to the studies of V.A. Romodanovskaja on fragments from Latin Vulgata, translated for the Gennadij's Bible of 1499. Her editions of Old Russian translations of the treaty *Rationale divinorum officiorum*…, written by Wilhelm Durand, and quotations from Lactantius2 should also be mentioned. Among others, the studies of E.V. Bodnarčuk3 and E.R. Skvairs4 on the Old-Russian translation of *Dyaloghus de Vite et Mortis* (*Prenie života so smert' ju*) occupy an important place. Nowadays N.A. Ziablincyna studies the translation of the anti-heretic treatise *Rationes breves magni rabi Samuelis iudaei nati* (*Učitelya Samuila oblichetiye*)5 . E.S. Fedorova analyzed the translation of *Contra perfideam judeorum* (*Protiv kovarstva iudeyev*) by Nicholas de Lyra. The translation of antiheretical treatise *Contra haereticos et gentiles*… (*Prenie Afanasija s Ariem*) is less studied.

The main purpose of this paper is to present the most relevant results of my complex study about the obtained copies of the Old Russian translation of the First book of Pomponius Mela's *Cosmographia, sive De Situ Orbis*, known also as *De Chorographia* – *Geografija* in Russian scientific tradition. This text was also translated into Old-Russian at the same epoque. All preserved and detected copies of this translation have been carefully studied by me both from an archaeographic and from a substantive point of view6 .

## 2. Pomponius Mela's *Cosmographia* in the 15th century Europe and in Russia

The text of the First book of *Cosmographia* is a brilliant compilation of known facts about Europe, Asia, and Africa in the Ancient world. It provides information about the topography, nature and important places of the described lands, as well as the habits and customs of native peoples. In his work, Mela mentions ancient gods (Zeus, Diana, Apollo, Neptune) and heroes (Anaximander, Alexander the Great among others) and retells some of the myths of antiquity.

Garzaniti 2008; Matasova 2014; Gardzaniti 2015; Matasova 2015; Pljuchanova 2017; Garzaniti 2019; and others.

Pomponius Mela's *Cosmographia* was one the most cherished ancient texts by Renaissance humanists and scholars. The first time the text was published in 1471 in Milan by the famous typographer Panfilo Castaldi. It was the first among many printed editions of this work on the Apennine Peninsula. During the second half of the 15th century there were at least nine other editions of *Cosmographia*. Seven of them were published in Venice (twice in 1477, twice in 1478, in 1482, in 1495, and in 1498), and two in Spain (Valencia 1982, Salamanca 1498). A beautiful map of the world was attached in the Venetian edition of 1482, and the 1498 edition was prefaced by a dedication to Pope Alexander VI, written by the humanist Hermolaus Barbarus.

Until the beginning of the 16thcentury humanists considered the text of Pomponius Mela to be the most complete and accurate description of oecumene. Even after the discoveries of Columbus and the realization that the ancient information about the world order had become invalid, Mela's text was still republished as an example of the excellent ancient Latin, the ideal with which the humanists sought to comply. During the first half of the 16th century this work was published at least 14 times (in Paris, Basel, Florence, and Venice). Among the publishers we can identify some of the most prominent typographers, distinguished innovators, and 'masters of the art of printing' of that time – Erhard Ratdolt, Simeon Bevilacqua, Joachim Vadianus, and Gilles de Gourmont.

At the same time Mela's wonderful text became known in Russia. Two copies of the Old Russian translation of the First book of Mela's *Cosmographia* were discovered at the end of 19th century by the famous Russian philologist professor A.I. Sobolevskij7 . The first copy – *M* – was made at the turn of the 15th and 16th centuries and it probably comes from *Posolskij prikaz* – the predecessor of the Ministry of Foreign Affairs8 . The second copy – *C* – was composed in the 17th century and was originally kept in the library of the Monastery of the Miracle of Archangel Michael at Chonae (*Čudov monastyr'*) in the Moscow Kremlin9 .

In 2014-2015 three more copies of this book were discovered. One of them – *S* – was found by O.L. Novikova10. It comes from the library of the Solovetsky Monastery and dates back to the late 15th century. It is kept now in the Russian National library in St. Perersburg11. Two others – discovered by K.V. Veršinin12 – can be referred to the second half of the 16th century. These originated from

8 Russian State Archive of the Ancient Acts in Moscow (*Rossijskij gosudarstvennyj arxiv drevnix aktov*, RGADA), Fund 181 *Manuscript collection of the archive of the Ministry of Foreign Affairs of the Russian Empire* (*Rukopisnyj otdel Moskovskogo glavnogo axiva Ministerstva inostrannyx del*, RO MGAMID). Reg. 1. Part 6. N. 514. Ff. 10v-40v.

9 Manuscript section of the State Historical Museum in Moscow (*Otdel rukopisej Gosudarstvennogo istoričeskogo muzeja*, OR GIM), *Čudovskoe sobranie* 347. Ff. 1v-16.

7 Sobolevskij 1903: 52-53.

10 Novikova 2015.

the private collections of ancient manuscripts of the late 19th century – that of Еgor Еgorov13 (*E*) and the other of Timofey Bolšakov14 (*B*). Now these manuscripts are kept in the Russian State Library. At present it is unknown where these manuscripts come from and how they appeared in these private collections. It can be mentioned only that the marginal notes in *E* give evidence of the fact that earlier the codex was used in the Northern parts of Russia (*Ust-Sysolsk* – modern Syktyvkar – is mentioned on the margins of the manuscript15).

## 3. Comparative reading of the obtained copies of Pomponius Mela's translation

My profound comparison of all five copies of the Old Russian translations of Pomponius Mela's *Cosmographia* revealed only insignificant differences between them. These differences are due to spelling variability16 and punctuation and substitution of single words by synonyms17. Sometimes Russian copyists did not understand some details and consequently made mistakes in spelling18.

It could be possible that there were more copies than we have obtained thus far. The following discrepancies (variants of the text) are proof of this fact. There are the words *Лимерикъ град по берегу ж* in copies *E* (f. 282), *B* (f. 379), and *C* (f. 15v). These words are omitted in *M*. Copy *M* in its turn contains several phrases, which are absent in *C*19. Therefore copy *C* was made not from *M*. Copies *E* and *B* can't be prototypes of *C* because a number of small fragments (lines) of the text which are present in *C* (lines of prototype(s) *E* and *B*?) can't be found in copies *E* and *B*20. The comparison of *C* and *S* revealed minimal discrepancies between them, which might have occurred during

14 Manuscript section of Russian State Library (*Naučno-issledovatel'skij otdel rukopisej Rossijskoj gosudarstvennoj biblioteki*, NIOR RGB), Fund 37 (*Sobranie S.T. Bolšakova*) 16. Ff. 371-379v.

13 Manuscript section of Russian State Library (*Naučno-issledovatel'skij otdel rukopisej Rossijskoj gosudarstvennoj biblioteki*, NIOR RGB), Fund 98 (*Sobranie E.E. Egorova*) 843. Ff. 265-282v.

15 F. 82v E.

16 E.g. *паки-пакы, пръво-перво, розливается-разливатца*, etc.

17 For example, *похораниваютъ* (f. 27v *М*) – *хоронят* (f. 10 *С*); *деля* (f. 13v *M*) – *ради* (f. 3 *C*); *зело* (f. 20v *M*) – *добре* (f. 6 *C*), etc.

18 E.g. *скотовъ* (f. 27v *M*) – *скотвов* (f. 10 *C*); *Меoтида* (f.13, 39v *M*) – *Меoдита* (f. 2v, 15v *C*); *Чермному морю* (Red sea) (f. 28v *M*) – *Черному* (f. 10v *C*); *почитаются* (f. 38v *M*) – *починаются* (f. 15 *C*); *Дономъ* (f. 12v *M*) – *Доломъ* (f. 266v *Е*); *Лаoдикиа* (f. 377 *B*) - *Лаодикия* (f. 11v *C*) – *Ладиoкиа* (f. 30v *M*); *Фитори* (f. 39v *M*) – *Фотори* (f. 379v *B*) – *Фотири* (f. 15v *C*), *Европиа* (f. 17v *M*) – *Еропиа* (f. 4 *S*), etc.

19 For example, in *M* there are words *оттоле море пакы шире чинится* (f. 36v), *изгыбаяся*

*потомъ великим лъбомъ* (f. 13v – 14), *Оутика и Карфагенъ, oба славныа грады* (f. 21). 20 In *Е* there are words which are lost in *M* and *C*: *роубежь. въ предних же было царем* (f. 271), *знають а иные и не знають женъ* (f. 273), *по городeКорытeскыи зракъ* (f. 277v). There are words added to the main text of *E* as marginal notes: *а инде глаголются. Таурикы а инде Москы* (f. 282) *и мужи же пешую брань сътворяют* (f. 282v). In *B* there are no words *а инде Москы. А инде. Амазоникы*, which are present in other copies: f. 39 *M*, 282 *Е*, 15 *C*.

copying21. It means that probably copy *S* in full (or another copy similar to it) could have been the prototype of *C*.

Copies *E* and *B* despite being read in codexes of similar content22, apparently have different prototypes*.* Thus there are phrases in *B*, which are not present in *E*23. And in *E*, in turn, there is a phrase which is omitted in *B*24. Nevertheless it shouldn't be excluded that they could have been made from the same copy, taking into consideration that each copyist made the same mistakes.

Despite these discrepancies it is easy to notice that copies *S*, *B*, *E*, and *C* were made from similar copies: a number of words and proper names are similarly or closely conveyed in *S*, *E*, *B*, and sometimes in *C*, while in *M* they look different25. Particularly noteworthy are discrepancies in constructions with demonstrative pronoun *оно*, pointed out by Dr. O.L. Novikova on the basis of comparison of copies *M* and *S*. According to our observations these inconsistencies are present not only in *S*, but in *E*, *B*, and sometimes in *C*. This is important as readings in copies *S*, *E*, *B*, and *C* are closer to the Latin original, where adverbs *quondam, olim*, and *aliquando* are used26. However, there are differences in the spelling of proper names in *S*, *E*, *B*, and *C*, which can't be explained only by the variability of spelling27.

Copy *M* contains Cyrillic semi-uncial marginal notes of the middle (second half?) of the 16th century28 and shorthand in the 17th century29 (marginal notes are not written in the same handwriting as in *C*). In *E* there are marginal notes in the handwriting of the 15th and 17th centuries30. This indicates that some-

29 F. 28 M.

30 Ff. 279, 282, 282v E.

one looked through the manuscripts in the 16th and 17th centuries. It was not an accidental reading: as stated below there was profound thought involved.

We can now assert that copy *S* is not the oldest version in existence prior to 1490. This becomes evident from the fact that on page 10 of this copy the words *и инаа чюднаа дeла* are written and crossed out with cinnabar. The same words can be found in their proper place in copy *S* and in all other copies31. In other words, the copyist of *S* accidently glanced at the wrong fragment of the prototypes and mistakenly included it in his work. The error was noticed later and unnecessary words were crossed out. There are more similar examples in copy *S*.

Copy *M* was not the oldest as well. It becomes evident from the already mentioned absence of the line (which is present in copies *E*, *B*, and *C*) that the copyist of the beginning of the 16th century had overlooked it. This gives additional evidence that the Old Russian translation was composed at the end of the 15th century (the dating of copy *S* is proof of that). Moreover some obvious slips of the pen in manuscript *M* can be considered as typical of the process of copying and not of translation32. More likely the prototypes of *E*, *B*, and *C* were not the oldest copies: as already mentioned above, some phrases, which are present in *M*, are absent in these copies.

Hence, minor but existing differences between all the copies allow to conclude that each of them might have had its own prototype. The oldest copy might have been the unpreserved prototype *S*, but it is hard to say for sure because of the defective nature of *S*33.

Consequently, it may be suspected the existence of not less than ten copies of the Old Russian translation of the First book of *Cosmographia* by Pomponius Mela in the 16th-17th centuries. Five of these copies have survived. The oldest copies date back to the last decade, or even the last quarter of the 15th century.

### 4. The problem of the Latin original and a hypothesis about the author

Thus, Mela's text appeared in Russia almost immediately after it became known among the humanists. A number of obtained and probable copies indicate that Mela's text was popular among Russian scribes of that time. They, just as the humanists had also become involved in the process of the comprehension of ancient manuscripts.

It is tempting to assume that Mela's text was translated from one of the incunables34. At first glance this seems very likely as researchers have a great variety of evidence at their disposal, which testifies that foreigners brought incunables to Russia. Both the treatise by W. Durand and *Rationes breves magni rabi Samu-*

31 F. 26v M; f. 10 S; f. 274 E; f. 376 B; f. 9 v C.

32 E.g. *И бе въ брани падение много от обеих от обоих странъ* (f. 22 *M*).

33 Novikova 2015: 37.

34 Romodanovskaja 2005: 594.

*elis iudaei nati*, as well as *Dyaloghus de Vite e Mortis* and *Historia destructionis Troiae (Troyanskaya istoriya)*, and some other texts were translated from incunables35. However, the situation with the First book of Mela's *Cosmographia* is different. The comparison of the Old Russian translation with the texts of incunables reveals that in the former there are omissions of fragments, which are present in incunables. It should be noted that we have the translation into Old Russian *only* of the First book of *Cosmographia,* whereas in all editions Mela's works are published in full.

This may indicate that the Old Russian translation was made not from any incunable, but from a manuscript. An important argument in favour of my hypothesis is the peculiarity of the transmission of proper names noticed by me. For instance, the city of Sida is translated *Сикла* – "Sicla"; as it is well known, in the Latin manuscript *d* resembled *cl*. This is one of the most frequent mistakes of Latin copyists, while in incunables letters are distinct and it is impossible to make such a mistake. This confirms the hypothesis that the translation was made from a manuscript. There is a similar situation with the name of the city Ocstros, which is translated as *Дестрос* – "Destros" and in some cases *Дествос* – "Destvos". Here *oc* is read as *de*, and in the second case *r* as *v*. This too is a typical mistake. The name of "gamphasantes" is translated *ганифасаде* – "ganifasade" (*m* turned into *ni*), and the people called "antibarani" has been translated by a scribe as *антибазане* – "antibazani" (*r* resembles *z*, this too is a frequent mistake).

What manuscript was it? Currently it has not been found and it is possible to assume that the manuscript was lost. Apparently, it was a copy of the 15th century made from an ancient manuscript. How could it find its way to Moscow? With high probability it may be assumed that it came with the books which Zoe Palaiologina might have brought to Moscow in 1472. It is important to specify that these books *were not* a part of the library of Byzantine emperors36 (the lost or mythological collection of Greek and Latin books widely known later as the "ancient library of Ivan the Terrible"37). These books could be a small collection of 15th century copies of ancient manuscripts made by Greek scribes or merchants: Cardinal Bessarion could have given it to Zoe as a dowry. Bessarion was an experienced bibliophile who devoted all his energy to the preservation and distribution of Ancient Greek culture in the Renaissance world38. What is more important – he was almost the only authoritative person in the West, who worried about the fate of the Greek world after 1453 and Zoe's destiny in particular.

Who was the translator of the manuscript? There is an abundance of Grecisms (*аравес, вактри, вретанииского, Камвиск царь, Кимон, Кизик, Селевкия, Олимпос, Омирос, Трацыус,* etc.). There is also an "Italian accent" in translation of some proper names. For example, *Certasor* is translated as *Чрътасор* (*ce*

35 *Ibid*: 593-594.

36 Fonkič 1977: 221-222.

37 Tichomirov 1968: 287.

38 Vast 1878; Bianca 2004; Mioni 2004; Ronchey 2006.

was read as *че*, not as *це*)*,* and *Damascena* is translated as *Дарамшена* (*sce* was read as *ше*, not as *сце*). All these particularities make it possible to assume that the Greeks who spoke Italian could be involved in the process of translation. Members of the Tarchaniota family are widely known in Russia (brothers Giorgio – *Yuri* and Dmitriy and Dmitriy's sons – Manuel39 and *Yuri Maloy* – Giorgio Minor40) as translators. They translated many texts from Latin in Novgorod. They spoke Italian and they were also directly related to the connections of the Russian state with Milan, Venice, and Rome. The Tarchaniotas also participated in the close circle (*dvor*) of Grand Duchess Zoe Palaiologina. Unfortunately, we don't know the precise name of the translator.

## 5. Perception of Mela's information in Russia

Pomponius Mela was a pagan author; he provided vivid and detailed description of pagan rites of the Ancient world, sanctuaries of gods, some fact of the deification of nature, etc. How did Russian scribes perceive this "aggressively pagan" information?

The perception of Mela's information in Russia and in the West was drastically different. In Muscovy the information of the ancient geographer was conceived not as 'objective' information about the world, but in the traditional manner of pursuit of biblical analogy.

Mela's text in Old-Russian codexes adjoins theological works and extracts from the Bible. But more importantly the infrequent marginal notes in the copies indicate that intellectuals tried to correlate the facts about pagan culture, about pagan way of mind, provided by Mela with the Bible. It was a traditional providential manner of understanding of the world order and of history, characteristic of Russian medieval intellectuals.

It is important to examine a notable marginal note "Psalm" in *M* made by a 16th century reader beside the description of pagan Egypt. This description is one of the most colorful fragments of the text. Here is the Latin variant of the fragment:

Terra expers imbrium mire tamen fertilis et hominum aliorumque animalium perfecunda generatrix. Nilus efficit, amnium in Nostrum mare permeantium maximus. […] non pererrat autem tantum eam, sed aestivo sidere exundans etiam irrigat, adeo efficacibus aquis ad generandum alendumque, ut praeter id quod scatet piscibus, quod hippopotamos crocodilosque vastas beluas gignit, glaebis etiam infundat animas ex ipsaque humo vitalia effingat. hoc eo manifestum est, quod, ubi sedavit diluvia ac se sibi reddidit, per umentes campos

39 For a long time, this Manuel was unknown by the researchers. But now we can affirm that he surely existed. Cf.: Vorob'jev, Matasova 2017; Matasova 2018.

40 Florja 1982.

quaedam nondum perfecta animalia, sed tum primum accipientia spiritum et ex parte iam formata, ex parte adhuc terrena visuntur41.

And here is its Old Russian translation:

Земля Египта велми родима и на человеческий род, и на скотъ: Нилъ еа поливаеть. Нилъ же река […] есть боле всех рекъ, иже в Наше Море вливается […] Имееть ж водоу родимоу не токмо на всякоую рыбоу, но и потами ражаеть, иже тлъкоутьс речнии кони, и коркодили ражаеть, иные многые скоты. Еще вода его въ земленую кромоу дыхание сътваряеть. И сътваряеть от земли живоущаа, то же явно есть, егда бо оубывая сливается с поль и въ своа берегы вълиется. Находять по полемъ некыа скоты еще не свръшена, но почати образитися, иная ж часть образна телесна, а инаа еще земля42.

In a number of Psalms we can find fragments that possess an extremely close resemblance to Mela's description of Egypt. Mela talks of the fertile soil of Egypt and about the very good life of all the animals there. And in Psalm 104 we can see a similar idea. Our Lord blesses water and soil and every animal is happy: our Lord "makes springs gush forth in the valleys; they flow between the hills; they give drink to every beast of the field…" (Ps. 104: 10-11). Then the psalmist exclaims:

O Lord, how manifold are your works! In wisdom have you made them all; the earth is full of your creatures. Here is the sea, great and wide, which teems with creatures innumerable, living things both small and great (Ps. 104, 24-25)

Surprisingly close to Mela's narrative on Egypt is a fragment of the biblical text; the Third book of Ezra, translated into Old Russian at the turn of 15th-16th centuries – at the same time with Mela43. Ezra writes:

Upon the fifth day thou said unto the seventh part, where the waters were gathered that it should bring forth living creatures, fowls and fishes: and so it came to pass. For the dumb water and without life brought forth living things at the commandment of God, that all people might praise thy wondrous works. Then did thou ordain two living creatures… (Ezra 3, 6: 47-49).

Thus, Mela describes how the water of the Nile gives life to the soil and it revives the animals. The Third book of Ezra tells how Our Lord blesses the water and this water gives life to animals as well!

Interestingly enough, in the first third of the 16th century, the Russian scribe Fedor Karpov asked Maksim the Greek about the meaning of these exact words of Ezra. The question arises – was Fedor Karpov one of the first readers of the Old Russian translation of Pomponius Mela's *Cosmographia*?

41 Parroni, 1984: 119-120.

42 Ff. 24v-25 *M*.

43 Romodanovskaya 2000: 6.

#### TATIANA MATASOVA

Another remarkable fragment in the description of Egypt in the Old Russian translation of Mela is dedicated to Apis – a holy bull for the Egyptians: "Apis populorum omnium numen est"44. Surely, this Apis was unknown in Russia. The translator didn't understand that in this particular case "apis" was a proper name and decided to translate "Apis" from Latin into Old Russian. In this way Apis suddenly became a bee (*пчела*): "Все же родове Египетьскыа чтят пчелоу акы божественоу"45. This example discovers that the Old Russian translator understood far from everything in the text. But later in the translation a providential interpretation is again revealed. After this phrase in Latin, Apis is marked only as "bos" ("вол"): "*bos* niger certis maculis insignis et cauda linguaque dissimilis aliorum. raro nascitur nec coitu pecudis, ut aiunt, sed divinitus et *caelesti* igne conceptus"46.

Here is this fragment in Old Russian:

Когда рождается волъ чернъ з белыми пестринами языкъ же оу него и хвостъ рознымъ подобиемъ, иже оу них редко ражаются, и глаголютъ тако: не от скотьска естьства зачать, но от божественаго огня47.

In Psalm 104 it is told that Our Lord "makes winds his messengers, and flames of fire his servants" (Ps. 104:4). It means that Our Lord makes fire to serve Him, and He can present Himself to people as fire. The medieval Russian intellectual – the reader of the translation of *Cosmographia* – as if accepting Egyptian perception of the divine nature of the fire, by which the bull might be conceived, implied a providential meaning of these words.

Mela also tells about the springheads of the Nile: the river

… crescit porro, sive quod solutae magnis aestibus nives ex immanibus Aethiopiae iugis largius quam ripis accipi queant defluunt, sive quod sol, hieme terris propior et ob id fontem eius minuens, tunc altius abit sinitque integrum…

In Old Russian this fragment looks like this:

Прибываеть же Нилъ и выливается или снегы тают иже на великых горах Ефиопьскых или о тоу пороу на веръховия его дожди великыя бывають. Инии же глаголютъ пескомъ оустья своя заносить. и от того прибываеть или пакы собою прибываеть оубываеть48.

Thus, Russian medieval intellectuals had a considerable knowledge about Egypt and – as we can suggest – wanted to know more about Egyptian nature. The comments of Maksim the Greek (in his *Skazaniya otčasti nedoumennyh nekiih rečenii v Slove Grigoria Bogoslova***-**Maksim the Greek's *Comments on St. Gregory the Theolo-*

44 Parroni, 1984: 121.

45 F. 27v *M*.

46 Parroni 1984: 121.

47 Ff. 27v-28 *M* It is interesting that in the Old Russian translation the word "divine" ("божественный") is used, while in the Latin original is used "heavenly" ("caelesti").

48 Ff. 25 об.-26 *M*.

*gian*) about the Nile and the fertility of the soil of the Nile banks are widely known49. Thus, these ideas exclusively relate to theology: Maximus the Greek tells about it in his exclusively theological work. The salvation of the soul and providential way of understanding the world order was the only interest of Orthodox intellectuals. It is time to mention, that in the middle of the 16th century there was a famous discussion between G.B. Ramusio and G. Fracastoro about the springheads of the Nile50. But it can be supposed that the Russian interest in the Nile and the comments of Maximus the Greek were not connected with this humanistic discussion51.

#### 6. Conclusions

The theological interpretation of Mela's text in Muscovy is vivid evidence that the Russian culture of those times was still (as it had been for a long time prior) focused on Orthodox doctrine and tried to dwell even on Renaissance subjects using the Bible.

Thus, rigorous scrutiny of the remaining copies of the Old Russian translation of the First book of Pomponius Mela's *Cosmographia* reveals that those texts which were important to the culture and thought of the Renaissance made their way into Russia and aroused a vibrant interest among scribes. However, the history of the appearance and treatment of Mela's text in Muscovy gives evidence of the fact that the perception of Renaissance traditions in the Russian world was imbued with a superficial quality. Nevertheless, the encounter and interaction with Renaissance traditions gave the Russian scribes a powerful impulse to examine and ponder the outside world and played its part in the development of the fundamental pillar of Russian culture-Orthodox theology. Nevertheless, the analyzed material reveals that the acquaintance of Russian scribes with the intellectual traditions of the Renaissance played a significant role in the formation of Russian culture at the time in question.

In conclusion, I would like to draw the attention of the field to my recent publication of a scientifically annotated Old Russian translation of the First Book of Pomponius Mela's *Cosmographia*52*.* It is my conviction and hope that increased interest in this important text on the part of European scholars will lead to new and exciting discoveries.

*degli Slavi: Incontri e dialoghi tra culture: Studi in onore di Giovanna Brogi Bercoff,* Firenze 2008, pp. 173-183.

Matasova 2016b T.A. Matasova, *Sof ' ja Paleolog,* Moskva 2016.

Matasova 2018 T.A. Matasova, *Nil Grek (Trachaniot), ep. Tverskoj*, in: *Pravoslavnaja enciklopedija, LI,* M. 2018, pp. 164-166.

## Abstract

The article deals with the results of the analysis of the Old Russian translation of the First book of *Cosmographia* by Pompons Mela. Mela's *Cosmographia* was admired and praised by humanists. The research of the way the text was comprehended and interpreted in Muscovy demonstrates the original features of the perception of the Renaissance traditions, ideas and values by Russian intellectuals. The study reveals that the comprehension of Mela's information was characterized by traditional manner of pursuit of biblical analogy. Thus, even the close acquaintance with the Renaissance culture did not change the essence of the Russian Medieval Orthodox culture.

*Keywords:* Pomponius Mela, Old-Russian translation, Muscovy, Renaissance, Orthodox theology.


**2.2 Analysis**

Data points consisted of the number of mosquitoes from each of the three species caught in a given trap on a given date. The unit of trap-night is used to describe the number of traps over the number of nights used to survey for each site.

*1 All traps except BG Sentinel traps were baited with approximately five pounds of dry ice in a plastic cooler with a hole in the bottom. The BG Sentinel traps were baited with dry ice and a commercial attractant (BG lure). 2 Data were transformed as the square root of (x + 0.1). The means in the lower position of each couplet are the backtransformed means of the transformed data. When means in a column are followed by the same letter, the means of transformed data are not statistically different (Tukey's mean separation test; alpha = 0.05).*

#### **Table 1.**

*Arithmetic mean (upper value of each couplet) and back transformed mean of transformed trapping rates of three abundant mosquito species caught in three types of trap in southern Missouri (2016–2018).*

*1 Data were transformed as the square root of (x + 0.1). Statistical analysis was done on transformed data but the means reported here are the arithmetic means of the original data and the back-transformed means of the transformed data. Means of transformed data (represented by backtransformed means) within each column followed by the same lower case letter are not significantly different (Tukey's mean separation test; alpha = 0.05).*

#### **Table 2.**

*Arithmetic mean (upper value of each couplet) and back-transformed mean of trapping rates (mosquitoes/ trap-night) for three species caught in CO2-baited CDC traps in four ecoregions of southern Missouri (summer, 2016–2018). The value in the lower position of each couplet is the back-transformed mean of the transformed data for trapping rate.*

## *Survey for a Vector of Zika Virus and Two Other Mosquito Species in Four Ecoregions… DOI: http://dx.doi.org/10.5772/intechopen.94499*

The survey consisted of a total of 653 trap-nights. The number of trap-nights for each trap type and ecoregion combination is reported in **Tables 1**–**4**. The data did not display a normal distribution and many data points reflected no catch ("0"); therefore, all data were transformed by taking the square root of (x + 0.1) where x was the number of mosquitoes of a given species caught in a single trap. This transformation appeared to improve the distribution of the data. For instance, the non-transformed data for *Ae. albopictus* caught in the CDC light trap had a skewness score of 2.92 and a kurtosis score of 11.23. After transformation, those scores were reduced to 1.21 and 1.01, respectively. Both transformed scores are below the recommended maximum thresholds recommended by West [11]. We used transformed data for all subsequent analyses and reported the results with back transformed

*1 Data were transformed as the square root of (X + 0.1). Statistical analysis was done on transformed data but the means reported here are the arithmetic means of the original data and the back-transformed means of the transformed data. Means of transformed data within each column followed by the same lower case letter are not significantly different (Tukey's mean separation test; alpha = 0.05).*

#### **Table 3.**

*Arithmetic mean (upper value of each couplet) and back-transformed mean of trapping rates (mosquitoes/ trap-night) for three species caught in CO2-baited fay-prince omnidirectional traps in four ecoregions of southern Missouri (summer, 2016–2018). The value in the lower position of each couplet is the back-transformed mean of the transformed data.*

*1 Data were transformed as the square root of (X + 0.1). Statistical analysis was done on transformed data but the means reported here are the arithmetic means of the original data and the back-transformed means of the transformed data. Means of transformed data within each column followed by the same lower case letter are not significantly different (Tukey's mean separation test; alpha = 0.05).*

*2 The BG Sentinel trap was not used in the Dissected Till Plans during any of the three years of the survey.*

#### **Table 4.**

*Arithmetic mean (upper value of each couplet) and back-transformed mean of trapping rates (mosquitoes/ trap-night) for three species caught in BG sentinel traps in three2 ecoregions of southern Missouri (summer, 2016–2018). The value in the lower position of each couplet is the back-transformed mean of the transformed data.*

means. Because trap catches for each trap type were significantly different for at least one species, we analyzed the trap catches by ecoregion separately for each trap type. We used the data for the most abundant species from each of three genera for the comparison: *Anopheles, Aedes* and *Culex*. These were also the three most abundant species in the entire survey regardless of genus. We calculated the mean trap catch for each species by ecoregion using an unbalanced analysis of variance in the PROC GLM of SAS, with mean separation using a Tukey's HSD test.

The Missouri Department of Health and Senior Services contracted this survey to the Master of Public Health Program and the Ozark Public Health Institute of Missouri State University in Springfield. (Contract #AOC16380144).


**5. Pregnancy and the history of sexual abuse**

Body memory can awaken when a woman's body starts to change during pregnancy. When the pregnancy becomes visible, this can be a sign that the body is no longer untouchable, that someone has already crossed the "line". Thus, a woman's gestational belly becomes part of the public arena, where everybody notices and sees it.  However, for a woman who has been sexually abused, her body becoming more noticeable and different can be extremely distressing. This is particularly the case if, for years and years, she had hidden every bodily change caused by the sexual abuse - even if only in her feelings and experiences.

Some studies have shown that girls who have been sexually abused in childhood are three times more likely to get pregnant before the age of 18 years [19] than those who have not.  According to other studies, pregnancy (as a consequence of sexual abuse) occurs in 11% to 20% of girls. Furthermore, more than 96% of underage prostitutes who have run away from home have been victims of sexual abuse in their childhood [9].

A study on a sample of 3,128 sexually abused underage girls showed that they engage more often in sexually risky behaviour (promiscuity, prostitution and pornography – seeking contact through sexuality). This study also reported that, compared to girls who had not been sexual abused, these girls were more likely to have had intercourse by the age of 15 years and more than one sexual partner. They were also less likely to use birth control during intercourse. Additionally, they more often used alcohol or other substances before having sexual inter‐ course. Among others, the following variables strongly stood out in this study: lack of parental supervision, presence of physical abuse, higher levels of school absenteeism, less involvement in extracurricular activities and lower grades (in comparison to those who had not been sexually abused). The difference between the two groups was statistically significant. The study also demonstrated that underage girls with a history of either sexual or physical abuse were twice as likely to become pregnant as teenagers, compared to girls without such a history. If they had experienced both sexual and physical abuse, they were four times as likely to get pregnant [21].

Seng et al. [22] found that women who had experienced PTSD during pregnancy were more susceptible to a spontaneous abortion, ectopic pregnancy and hyperemesis. Other studies report that sexually abused women have a statistically significant higher number of compli‐ cations during pregnancy. Among the most common are: bleeding, severe vomiting, x-ray or radiotherapy in the first semester, alcohol, smoking, medications, accidents, infectious diseases, threatened abortions with hospitalization and severe illness [23]. Some abused women report several health complications and a higher use of health care services during pregnancy. However, these women do not experience more obstetric complications during their pregnancies and deliveries [24].

 In general, children whose mothers were depressed during pregnancy show changes in their neurological functioning. They are more withdrawn, irritable and inconsolable than children whose mothers were not depressed during pregnancy [25]. Prenatal depression can lead to deficient care during pregnancy and bad eating habits, as well as the abuse of various substances like tobacco, alcohol and illegal drugs.

**Smoking** can lead to spontaneous abortion, as well as increasing the risk of an enlarged thyroid gland in children, low body weight and deformation.  The foetuses of women who chronically drank **alcohol** during pregnancy exhibited serious morphological (related to the form of the organism) and developmental abnormalities. Not all children exposed to alcohol during pregnancy suffer such serious conditions. However, there is a high probability that they will have neurological and cognitive disorders such as a lower reaction time and reduced attention spans.

**Using illegal drugs** during **pregnancy** is also problematic. Using **marihuana** during pregnancy can lead to a child having lower mental abilities, hyperactivity, impulsiveness, carelessness, inattention, delinquency and the externalizing of problems. **Cocaine abuse** during pregnancy is associated with a weakened, impaired processing of aural informa‐ tion, an increased risk of spontaneous abortion, premature labour pains, a stillborn child and microcephaly (the child having a small head). Heroine abuse and the use of other narcotics can cause a premature birth, the death of the foetus, the child's addiction, low body weight and cognitive and behavioural problems. **LSD and inhalants abuse** can be related to various deficits at birth [26].

In addition to drugs, women often silence their abused bodies with food. Waugh and Bulik [27] have found that women **with eating disorders during pregnancy** are more exposed to a caesarean section and have more problems in maintaining breastfeeding. Often, the disorders continue after the birth, leaving them at a high risk of developing postnatal depression.

Katarina Neff [28], a doula who has a lot of experience with pregnant women who have been sexually abused, believes that when a sexual abuse survivor with PTSD becomes pregnant, she may develop the following symptoms:

someone has already crossed the "line". Thus, a woman's gestational belly becomes part of the public arena, where everybody notices and sees it.  However, for a woman who has been sexually abused, her body becoming more noticeable and different can be extremely distressing. This is particularly the case if, for years and years, she had hidden every bodily change caused by

Some studies have shown that girls who have been sexually abused in childhood are three times more likely to get pregnant before the age of 18 years [19] than those who have not.  According to other studies, pregnancy (as a consequence of sexual abuse) occurs in 11% to 20% of girls. Furthermore, more than 96% of underage prostitutes who have run away from home have been victims of sexual abuse in

A study on a sample of 3,128 sexually abused underage girls showed that they engage more often in sexually risky behaviour (promiscuity, prostitution and pornography – seeking contact through sexuality). This study also reported that, compared to girls who had not been sexual abused, these girls were more likely to have had intercourse by the age of 15 years and more than one sexual partner. They were also less likely to use birth control during intercourse. Additionally, they more often used alcohol or other substances before having sexual inter‐ course. Among others, the following variables strongly stood out in this study: lack of parental supervision, presence of physical abuse, higher levels of school absenteeism, less involvement in extracurricular activities and lower grades (in comparison to those who had not been sexually abused). The difference between the two groups was statistically significant. The study also demonstrated that underage girls with a history of either sexual or physical abuse were twice as likely to become pregnant as teenagers, compared to girls without such a history. If they had experienced both sexual and physical abuse, they were four times as likely to get

Seng et al. [22] found that women who had experienced PTSD during pregnancy were more susceptible to a spontaneous abortion, ectopic pregnancy and hyperemesis. Other studies report that sexually abused women have a statistically significant higher number of compli‐ cations during pregnancy. Among the most common are: bleeding, severe vomiting, x-ray or radiotherapy in the first semester, alcohol, smoking, medications, accidents, infectious diseases, threatened abortions with hospitalization and severe illness [23]. Some abused women report several health complications and a higher use of health care services during pregnancy. However, these women do not experience more obstetric complications during

Women with a history of sexual abuse **experiencedepression in the prenatal and the postnatal period much more often** than women who have not been sexually abused. In general, children whose mothers were depressed during pregnancy show changes in their neurological functioning. They are more withdrawn, irritable and inconsolable than children whose mothers were not depressed during pregnancy [25]. Prenatal depression can lead to deficient

the sexual abuse - even if only in her feelings and experiences.

their childhood [9].

122 Sexology in Midwifery

pregnant [21].

their pregnancies and deliveries [24].

"Because birth is so different for every woman," comments Kristina, "women who have dealt with the same trauma may react in completely different ways and have completely different fears. Mostly, they are afraid of dealing with the first trauma again, regardless of if it was an abusive situation or a previous traumatic birth." There are some **specific fears that may manifest for a sexual abuse survivor with PTSD**, such as:

Sometimes the consequences of sexual abuse are not extensive or it does not seem that there are any. However, some women have been surprised and distressed by the feelings or memories that surface once they are pregnant, giving birth or mothering their child.

*"Until I got pregnant, I had no problems or any difficult memories of the sexual abuse I had suffered in my childhood. It seemed to me that those who said it was a serious trauma exaggerated, since my life was quite okay. I don't know what happened afterwards, but I can't even describe the degree of worry, fear and anxiety I started experiencing after I got pregnant, especially after I started showing. A terrifying fear that I'd lose the child because my body was dirty due to the abuse and the child couldn't develop in such conditions...sudden and severe anxiety attacks because I no longer had control over my body, my weight, my belly, which kept growing..."*

*"My pregnancy was nothing special until the moment the child began kicking in my belly. I was overwhelmed by the uncontrollable feelings of panic. Once, I even had to go to the ER. I felt guilty because I just wanted to pull the child right out, since every movement that was not under my control unnerved me intensely. At moments, I even wished for the child to die so that the distress would stop but I knew that, then, another would begin. I was somewhat appeased by being told I was having a girl because I realized that a boy would obviously remind me too much of the perpetrator."*

Clinical practice has shown that many pregnant women who have been sexually abused in the past often experience distress and new traumas in situations where women without a history of sexual abuse do not experience - or to a substantially smaller degree [29]. Even the usual gynaecological examinations can be very unpleasant due to the groping. In this case, it is very important that the gynaecologist is professional and sensitive. Nevertheless, when a woman is pregnant, carrying a developing and growing being, she is all the more sensitive to every touch and procedure related to her womanhood and her body [30].

*"The visit to the gynaecologist reminds me very much of rape.  I think that someone inserting their fingers in your vagina is a matter of sex and I can't imagine what else this action would be good for. I find the stirrups particularly horrible. But, on the other hand, I am also aroused. When I was at the gynaecologist's last year, I had the same feeling as after sex, only without the kissing and petting.  I was* *very much aroused when I went home and I was quite ashamed. I felt disgusted with myself and wished that someone would actually rape me. I'd pay him to go to bed with me and treat me really nastily, like his slave."*

*"It's the most horrible doctor's examination in the world. I go there when they send me the third invitation because I have to have regular check-ups.  The thought that I will have to go there almost every month when I'm pregnant makes me not want to ever get pregnant."*

*"What bothered me most was that some people touched my baby bump without permission. There were a few times when I had to leave the company and go to the toilet to vomit because I was so disgusted with the touching and, at the same time, angry with myself that I didn't draw the line and say I didn't like it."*

Post-traumatic effects of sexual abuse can be a big problem for survivors and a source of great fear. This is due to the subconscious triggering of traumatic memories during pregnancy (prenatal care) and delivery (the experience of birth). Women may avoid necessary medical care because of these fears. Furthermore, they may be so devastated by their experiences that they have difficulty enjoying and caring for their newborns. With this in mind, it can be highly beneficial if the personnel (midwifes, doctors, doulas, etc.) that a woman encounters during pregnancy, childbirth and the postnatal period are acquainted with her trauma and react appropriately. This ensures an even greater feeling of safety and support for the traumatized woman. Compassion in such hard times accelerates the healing of the reawakened trauma. At the same time, unkindness, roughness and unprofessionalism re-traumatize the pregnant woman and cause her additional distress and pain [29].


**4. Body fatness**

Body fatness and abdominal obesity is normally estimated by body mass index (BMI), waist circumference, and waist-to-hip ratio [7]. There is strong convincing evidence that body fatness increases the risk of colorectal, colon, and rectum cancer [7]. However, cognizance should be taken of the fact that these anthropometric measurements have limitations as they do not distinguish between lean and fat mass [8]. Evidence supporting a clear dose-response association was related to showing a significant increased risk of CRC with an increased BMI [8, 43, 44]. There is evidence of a nonlinear dose response, whereby the increased risk is higher at a BMI beyond 27 kg/m2 for CRC. Significant positive associations were observed for CRC in the dose-response analysis for waist circumference and waist-to-hip ratio [8]; hence, the level of evidence is being referred to as convincing for abdominal obesity [33]. In contrast to the vague findings regarding the role of individual nutrients or foods, the strong consistent association between obesity and CRC (at least in men)

further underscores the importance of combined integrated effects of nutrients/ foods over their individual effects. These effects probably do not only reflect the imbalance between energy intake and expenditure but the often suboptimal quality of the diet associated with the development of obesity [4].


Meet the editor

Reda R. Gharieb PhD, is Professor of Biomedical and Bioelectronics Engineering at Assiut University, Egypt. He has worked for Fairway Medical Technologies Inc. and Seno Medical Instruments Inc., Texas, USA, on their photoacoustic imaging (PAI) technology. He has developed algorithms for 2D image reconstruction in PAI of breast and prostate cancers. He also has developed an algorithm for 3D image reconstruction in PAI of

a small animal, using a rotated ARC-shaped sensor array. Dr. Gharieb has collaborated with brilliant international professors, scholars and engineers from academic and industrial institutions. He authored two books, three chapters, two patents and about 60 papers. His research interests include signal/image processing, modeling and simulation, machine learning, statistical and scientific computing, neuro engineering, bioelectronics and bioinstrumentation.

Contents

**Section 1**

*by Reda R. Gharieb*

*by Chin Hsia*

**Section 2**

*by Surya Narayan Thakur*

of the Acoustic Signal

Photoacoustic Imaging of the Eye *by Yanxiu Li and Yannis Mantas Paulus*

for Medical Ultrasonic Imaging Applications

**Preface III**

Photoacoustic Imaging: Principles and Advances **1**

**Chapter 1 3**

**Chapter 2 11**

**Chapter 3 33**

**Chapter 4 55**

Photoacoustic Imagine: Applications **65**

**Chapter 5 67**

**Chapter 6 91**

Photoacoustic Imaging for Cancer Diagnosis: A Breast Tumor Example

A Balanced Slew-Rate High-Voltage Integrated Bipolar Pulse Generator

Determination of the Source Localization and the Beginning Time

*by Rostyslav Romanyshyn, Galyna Romanyshyn and Igor Romanyshyn*

Photoacoustic Imaging in Gastroenterology: Advances and Needs *by Sheena Bhushan, Sharmila Anandasabapathy and Elena Petrova*

Photoacoustic Spectroscopy of Gaseous and Condensed Matter

## Contents

Preface

Photoacoustic imaging (PAI) is an emerging non-invasive imaging modality that integrates the advantages of deep ultrasound penetration and high optical contrast. The fundament of PAI is photoacoustic effect, which takes place when a lightabsorbing material sample is irradiated by a laser pulse, causing temporal changes of the temperature at loci where light energy is absorbed. Due to this temperature change, the region where light energy is absorbed exhibits transient thermoplastic expansion, generating a pressure pulse. This pressure pulse propagates within the sample body to the surrounding areas and is detected by a set of ultrasonic sensors. The amplitude, time of arrival at the sensor, and duration of each detected pulse are proportional to the absorption coefficient, the location, and the size of the vibrating region, respectively. The set of detected ultrasonic signals are used by different image reconstruction algorithms to produce an image that maps the spatial distribution of the light absorption coefficient within the material sample. Because it is nonionizing, nondestructive, and high-resolution, PAI is opening new frontiers in medical imaging. It provides diagnostic imaging for the anatomy and physiology of small animals, anatomical tissue/organ structure, oxygenation status, blood flow, abnormal morphology, functional vasculature characteristics, skin burns,

hypermetabolism, and vulnerable plaques in the coronary arteries.

to visualize retinal metabolism and melanin of the eye.

community.

This book, *Photoacoustic Imaging: Principles, Advances and Applications*, provides interested readers with the principle knowledges, advanced methodologies, and new applications associated with PAI technology. The first section, which contains four chapters, covers the generation and detection of photoacoustic signals, sound source localization, and image reconstruction and formation. This section cites different research papers on emerging deep learning-based image reconstruction. In the second section, which contains two chapters, presents relatively new diagnostic PAI applications. The first chapter discusses PAI application in gastroenterology. It explores the clinical use of PAI-based endoscopy in the diagnosis of esophageal cancer, inflammatory bowel diseases, and pancreatic cancer. The final chapter reports that many eye diseases, including macular degeneration and diabetic retinopathy, involve abnormalities in the vasculature. Therefore, PAI for visualizing and investigating the vasculature can be incredibly helpful as a diagnostic tool for eye diseases. These include iris vasculature, corneal neovascularization, retinal vasculature, choroidal vasculature, and retinal neovascularization. PAI is also used

We believe that the applications of PAI in gastroenterology and eye diseases are relatively new and deserve attention. In the future, there is sure to be more literature on using PAI in other areas. We hope this book serves as a channel for exchanging perspectives, experiences, and research results within the scientific

Finally, the editor acknowledges and appreciates the effort and kind cooperation of all the chapter authors and their willingness to exchange their experiences and research outcomes with other colleagues. The editor also acknowledges and appreciates the important editing and reviewing services, strong support, and kind


**3. Improvement of** *in vitro* **protein digestibility**

The main goal of experiments on silencing of kafirin genes is to improve seed storage protein digestibility. Herewith, depending on the structure of the genetic construct, suppression of certain subclasses of kafirins, and the cultivars used in experiments, the level of digestibility varied significantly.

For example, transgenic plants of cv. Tx430 carrying the ABS166 genetic construct containing inverted repeats of several kafirin genes (α, γ, δ) separated by the intron sequence of the alcohol dehydrogenase gene (*ADH1*) and controlled by the 19-kDa α-zein promoter from maize were characterized by improved *in vitro* protein digestibility. Pepsin treatment of the raw flour and flour that underwent the cooking procedure resulted in 78% and 61% digestibility, respectively, while in the non-transgenic control these indicators varied within 40–50% and 34–40%, respectively [34, 35]. The genetic construct for the silencing of δ- and γ-kafirins (ABS149) also improved the digestibility of raw flour, but did not affect the digestibility of the cooked flour.

Subsequently, new transgenic plants were obtained in the sorghum public line P898012 using other genetic constructs ABS042 and ABS044, created during the ABS (Africa Biofortified Sorghum) project [22]. In these plants, an improvement in the digestibility of flour subjected to the cooking procedure was recorded: from 28% in the control to 39% (for the ABS042 construct for silencing γ- and δ-kafirins), and up to 59% (for the ABS044 construct for silencing α-, γ- and δ-kafirins).

Analysis of ultrastructure of protein bodied showed that in transgenic lines with α-kafirin silencing protein bodies were irregular in shape and had invaginations similar to P721Q mutant [34, 35]. In transgenic lines with γ-kafirin silencing, a diameter of protein bodies was reduced in comparison with original non-transgenic line [36]. In addition, in one of the studied lines, 42–1, protein bodies were highly irregular in shape, with deep invaginations present at the periphery, while in the line 42–2, the protein bodies had small peripheral indentations that gave the boundary region a cracked appearance.

In the experiments of T. Kumar et al. [20] the genetic constructs pPTN915 and pPTN1017 designed for the induction of silencing γ- or α-kafirin, respectively, were also introduced into the genome of the Tx430 line through agrobacterial transformation. *In vitro* digestibility of proteins extracted from the flour of transgenic kernels with silencing of γ-kafirin, subjected to cooking procedure, did not differ from the non-transgenic control, while the silencing of α-kafirin by pPTN1017 improved the *in vitro* protein digestibility of flour subjected to cooking.

Transgenic plants of cv. Zh10 obtained in our experiments carrying the genetic construct pNRKAF for silencing *γ-KAFIRIN-1* gene, also had a significantly

*Grain and Seed Proteins Functionality*

**2. Decreased content of kafirins**

introducing a genetic construct capable to induce *γ-KAFIRIN-1* silencing. For silencing the *γ-KAFIRIN-1* gene we used the construct pNRKAF [23] that consisted of segment of its nucleotide sequence ([31], GeneBank accession no. M73688) in forward and inverted orientation, which was separated by the sequence of the maize *ubi1*-intron. This construct was driven by the *35S* promoter. Such a construct should suppress the expression of the *γ-KAFIRIN-1* gene using RNA interference. A decrease in the level of γ-kafirin should have "stripped" the protein bodies in

In this chapter, we describe phenotypic effects of RNAi-silencing of kafirin genes in two sorghum cultivars – Zheltozernoe-10 (Zh10) and Avans, which contain pNRKAF genetic construct introduced by agrobacterial transformation, as well as characteristic features of other sorghum lines carrying similar genetic constructs for

The primary effect of the functioning of genetic constructs for RNA silencing of kafirin genes is a decreased level of transcripts of these genes. Such an effect was shown for the pPTN915 genetic construct, designed to suppress the expression of

*SDS-PAGE of kafirins from kernels of transgenic plants from the T1 generation of the RNAi mutant, cv. Avans, isolated under reducing conditions (with the addition of 2-mercaptoethanol). 1 – Original non-transgenic cv. Avans; 2–7 – Individual plants from the T1 family: 2–6 – Plants with a floury endosperm, containing ubi1-intron; 7 – Plant with a vitreous endosperm, not containing ubi1-intron; M – Molecular mass markers. Kafirins were extracted according to [20]. The arrow marks* α*-kafirin; the dotted arrow marks* γ*-kafirin.*

transgenic plants and facilitated the digestion of α-kafirins.

silencing kafirin genes created by other research groups (**Table 1**).

**146**

**Figure 1.**

improved *in vitro* digestibility of flour proteins [22]. Comparison of electrophoretic spectra before and after pepsin digestion showed that in the transgenic plants the amount of undigested monomers of α-kafirin and total undigested protein was significantly less (1.7–1.9 times) than in the original non-transgenic line. The digestibility level reached 85.4%, while in the original line this value was about 60%. It is noteworthy that in the kernels of transgenic plant No. 94–3-08 (T2) with a thick vitreous endosperm, the differences in the digestion of kafirins were more pronounced: the amount of undigested monomers was 17.5 times less, and the amount of total undigested protein was 4.7 times less than in the original line, while the level of digestibility reached 92%.

Plants from the T3 generation inherited the improved digestibility of kafirins. In these plants, kernels had either a modified type of endosperm with reduced vitreous endosperm, or an endosperm with a well-defined vitreous layer. The level of digestibility of endosperm proteins in these plants was 83–90%, significantly higher than that of the original non-transgenic line (**Figure 2**). Apparently, a decrease in the level of γ-kafirin increases the digestibility of α-kafirins. This increase may be due to chemical reasons (decrease in the amount of polymers) and/or physical reasons (changes in the spatial arrangement of α-kafirins in protein bodies, which increase their availability for cleavage by pepsin). The effect of increased digestibility of kafirins was also observed in plants from the T4 generation; however, in some cases it disappeared, possibly due to the instability of the introduced genetic construct, or due to its silencing (see below).

After experiments with the model cv. Zh10, we set the task of obtaining RNAi mutants with improved digestibility of kafirins in the new commercial cultivar Avans, which is characterized by a number of agronomically valuable traits. The analysis of the *in vitro* digestibility of proteins from kernels that set on one of the transgenic plants (#1–1) obtained by *Agrobacterium*-mediated genetic transformation with the strain carrying pNRKAF genetic construct showed a significantly higher level of digestibility compared to the original non-transgenic cultivar (**Figure 3**) (93% vs. 57%, according

#### **Figure 2.**

*Electrophoretic spectra of proteins from the flour of transgenic plants from T3 family #94–3-08 with normal vitreous endosperm. 1–6 – Individual plants from T3 generation; 7, 8 – Original non-transgenic line Zh10. 1, 3, 5, 7 – Before, 2, 4, 6, 8 – After pepsin digestion. M - molecular mass markers (kDa). [23].*

**149**

**Figure 4.**

*silencing, and original cv. Avans (B).*

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage…*

to the densitometry of SDS-PAGE patterns). A high level of kafirin digestibility was

*Electrophoretic spectra of proteins from the flour of sorghum cv. Avans (1–3), transgenic plant #1–1 (4–6) and non-transgenic plants #5–1 (7–9) and #6–4 (10–12). M - molecular mass markers (kDa). 3, 6, 9, 12 – Before,*

An important consequence of the functioning of genetic constructs for silencing kafirin genes is a change in the texture of endosperm: in transgenic plants, in most cases, there is complete or partial loss of the vitreous layer, as a result of which the kernels contain only floury endosperm [21, 22, 34, 35]. In our experiments, the

*Cross sections of the kernels of the RNAi mutant #1-1 (A) carrying genetic construct pNRKAF for RNAi*

*DOI: http://dx.doi.org/10.5772/intechopen.96204*

observed also in the next generation, T1.

*1, 2, 4, 5, 7, 8, 10, 11 – After pepsin digestion.*

**Figure 3.**

**4. Modification of endosperm texture**

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage… DOI: http://dx.doi.org/10.5772/intechopen.96204*

#### **Figure 3.**

*Grain and Seed Proteins Functionality*

the level of digestibility reached 92%.

construct, or due to its silencing (see below).

improved *in vitro* digestibility of flour proteins [22]. Comparison of electrophoretic spectra before and after pepsin digestion showed that in the transgenic plants the amount of undigested monomers of α-kafirin and total undigested protein was significantly less (1.7–1.9 times) than in the original non-transgenic line. The digestibility level reached 85.4%, while in the original line this value was about 60%. It is noteworthy that in the kernels of transgenic plant No. 94–3-08 (T2) with a thick vitreous endosperm, the differences in the digestion of kafirins were more pronounced: the amount of undigested monomers was 17.5 times less, and the amount of total undigested protein was 4.7 times less than in the original line, while

Plants from the T3 generation inherited the improved digestibility of kafirins. In these plants, kernels had either a modified type of endosperm with reduced vitreous endosperm, or an endosperm with a well-defined vitreous layer. The level of digestibility of endosperm proteins in these plants was 83–90%, significantly higher than that of the original non-transgenic line (**Figure 2**). Apparently, a decrease in the level of γ-kafirin increases the digestibility of α-kafirins. This increase may be due to chemical reasons (decrease in the amount of polymers) and/or physical reasons (changes in the spatial arrangement of α-kafirins in protein bodies, which increase their availability for cleavage by pepsin). The effect of increased digestibility of kafirins was also observed in plants from the T4 generation; however, in some cases it disappeared, possibly due to the instability of the introduced genetic

After experiments with the model cv. Zh10, we set the task of obtaining RNAi mutants with improved digestibility of kafirins in the new commercial cultivar Avans, which is characterized by a number of agronomically valuable traits. The analysis of the *in vitro* digestibility of proteins from kernels that set on one of the transgenic plants (#1–1) obtained by *Agrobacterium*-mediated genetic transformation with the strain carrying pNRKAF genetic construct showed a significantly higher level of digestibility compared to the original non-transgenic cultivar (**Figure 3**) (93% vs. 57%, according

*Electrophoretic spectra of proteins from the flour of transgenic plants from T3 family #94–3-08 with normal vitreous endosperm. 1–6 – Individual plants from T3 generation; 7, 8 – Original non-transgenic line Zh10. 1, 3,*

*5, 7 – Before, 2, 4, 6, 8 – After pepsin digestion. M - molecular mass markers (kDa). [23].*

**148**

**Figure 2.**

*Electrophoretic spectra of proteins from the flour of sorghum cv. Avans (1–3), transgenic plant #1–1 (4–6) and non-transgenic plants #5–1 (7–9) and #6–4 (10–12). M - molecular mass markers (kDa). 3, 6, 9, 12 – Before, 1, 2, 4, 5, 7, 8, 10, 11 – After pepsin digestion.*

to the densitometry of SDS-PAGE patterns). A high level of kafirin digestibility was observed also in the next generation, T1.

#### **4. Modification of endosperm texture**

An important consequence of the functioning of genetic constructs for silencing kafirin genes is a change in the texture of endosperm: in transgenic plants, in most cases, there is complete or partial loss of the vitreous layer, as a result of which the kernels contain only floury endosperm [21, 22, 34, 35]. In our experiments, the

#### **Figure 4.**

*Cross sections of the kernels of the RNAi mutant #1-1 (A) carrying genetic construct pNRKAF for RNAi silencing, and original cv. Avans (B).*

RNAi mutant #1–1 of cv. Avans, had also a floury type of endosperm (**Figure 4**). It should be noted that, in similar experiments in maize, silencing of different zein genes also resulted in reduction of the vitreous endosperm and formation of kernels with floury endosperm [37–39]. It was shown that γ-zein gene plays an important role in the formation of the floury endosperm, and silencing of this gene modified the structure of protein bodies and their connection with starch grains that result in formation of floury endosperm [38].

Unfortunately, the presence of floury endosperm is a significant disadvantage of the obtained lines, since the absence of a vitreous layer increases the fragility of the kernels and reduces its resistance to fungal diseases. It should be noted that the floury (opaque) type of endosperm is characteristic of the P721Q mutant and many

#### **Figure 5.**

*Longitudinal sections of kernels of the original non-transgenic line Zheltozernoe-10 (A) and transgenic plants carrying pNRKAF genetic construct for RNA silencing of the γ-KAFIRIN-1 gene (B-F), differing in the degree of development of the vitreous endosperm. Vitreous endosperm is marked with white arrows.*

**151**

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage…*

breeding lines with improved digestibility of kafirins derived from it. Overcoming

In this regard, the transgenic plants of cv. Zh10 with the genetic construction pNRKAF for *γ-KAFIRIN-1* gene silencing are of special interest, since in most cases they had sectors or of the vitreous endosperm in the kernels, or the vitreous endosperm formed a continuous thin layer along the periphery of the kernels (**Figure 5**). It is noteworthy that the formation of the vitreous endosperm in such kernels did not reduce the digestibility of kafirins. Moreover, a plant (94–3-08) was found in T2, in whose progeny (T3, kernels is shown in **Figure 5F**) a high level of kafirin digestibility (88–90%) was combined with normal vitreous endosperm [23]. The fact of obtaining such plants shows that an increase in the digestibility of sorghum kafirins may not be associated with the reduction of the vitreous layer and formation of floury endosperm. Further investigation of these plants is needed to understand the role of the γ-kafirin in development of hard endosperm

Previously, transgenic plants with inclusions of vitreous endosperm surrounded by a floury endosperm were also observed in the transgenic plants of cv. Tx430, which contains a genetic construct for silencing α- and γ-kafirins [35]. At the same time, co-suppression of the δ-kafirin and γ-kafirin subclasses did not change the endosperm type in this cultivar. Apparently, the formation of different types of endosperm is due to the peculiarities of the expression of genetic constructs in the

In this regard, it should be noted that the nucleotide sequence that we used in the genetic construct pNRKAF was homologous not only to the γ-*KAFIRIN-1* gene located in the chromosome 2 of the sorghum genome but also to the locus of the chromosome 9 encoding bi-functional protease inhibitor protein (Pfam: PF00234) belonging to the LTP-family (lipid transfer proteins) [23]. It is possible that a higher kafirin digestibility in plant 94–3-08 and its progeny could be due mainly to the suppression of the synthesis of the protease inhibitor, which did not entail a change

These data indicate a possible effect of protease inhibitors on the digestibility of proteins in sorghum flour, which remains poorly understood. Purposeful designing of genetic constructs for RNA-silencing of protease inhibitors and their introduction into sorghum genome can help to obtain lines with improved digestibility of

An important consequence of silencing of the prolamine genes in cereals is an increase in the synthesis of other proteins, including those with a higher content of essential amino acids. For example, in transgenic maize plants with α-zein silencing, a double content of tryptophan and lysine was observed [40]. In rice, it was shown that silencing of 13 kDa prolamine increases the total lysine content up to 56% as a result of a compensatory increase in the synthesis of lysine-rich glutelin, globulins, and chaperones [41]. A significant increase in the lysine content (up to 3.3 g / 100 g of protein, compared to 2.1 g/100 g of protein in the non-transgenic control) was found in transgenic sorghum plants carrying complex genetic constructs for RNA silencing of kafirins (ABS032, ABS149) [35]. However, these genetic constructs carried, along with the fragments of the kafirin genes, the fragments of the lysine ketoglutarate reductase gene, which controls the catabolism of free lysine. This fact does not allow drawing a conclusion on the effect of kafirin silencing on the

kafirins, in which the endosperm could be of the usual vitreous type.

*DOI: http://dx.doi.org/10.5772/intechopen.96204*

in sorghum.

genome of the recipient line.

in the texture of the endosperm.

**5. Increased synthesis of other proteins**

increase in the lysine content in sorghum.

this correlation is an extremely difficult and urgent task [8].

#### *RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage… DOI: http://dx.doi.org/10.5772/intechopen.96204*

breeding lines with improved digestibility of kafirins derived from it. Overcoming this correlation is an extremely difficult and urgent task [8].

In this regard, the transgenic plants of cv. Zh10 with the genetic construction pNRKAF for *γ-KAFIRIN-1* gene silencing are of special interest, since in most cases they had sectors or of the vitreous endosperm in the kernels, or the vitreous endosperm formed a continuous thin layer along the periphery of the kernels (**Figure 5**). It is noteworthy that the formation of the vitreous endosperm in such kernels did not reduce the digestibility of kafirins. Moreover, a plant (94–3-08) was found in T2, in whose progeny (T3, kernels is shown in **Figure 5F**) a high level of kafirin digestibility (88–90%) was combined with normal vitreous endosperm [23]. The fact of obtaining such plants shows that an increase in the digestibility of sorghum kafirins may not be associated with the reduction of the vitreous layer and formation of floury endosperm. Further investigation of these plants is needed to understand the role of the γ-kafirin in development of hard endosperm in sorghum.

Previously, transgenic plants with inclusions of vitreous endosperm surrounded by a floury endosperm were also observed in the transgenic plants of cv. Tx430, which contains a genetic construct for silencing α- and γ-kafirins [35]. At the same time, co-suppression of the δ-kafirin and γ-kafirin subclasses did not change the endosperm type in this cultivar. Apparently, the formation of different types of endosperm is due to the peculiarities of the expression of genetic constructs in the genome of the recipient line.

In this regard, it should be noted that the nucleotide sequence that we used in the genetic construct pNRKAF was homologous not only to the γ-*KAFIRIN-1* gene located in the chromosome 2 of the sorghum genome but also to the locus of the chromosome 9 encoding bi-functional protease inhibitor protein (Pfam: PF00234) belonging to the LTP-family (lipid transfer proteins) [23]. It is possible that a higher kafirin digestibility in plant 94–3-08 and its progeny could be due mainly to the suppression of the synthesis of the protease inhibitor, which did not entail a change in the texture of the endosperm.

These data indicate a possible effect of protease inhibitors on the digestibility of proteins in sorghum flour, which remains poorly understood. Purposeful designing of genetic constructs for RNA-silencing of protease inhibitors and their introduction into sorghum genome can help to obtain lines with improved digestibility of kafirins, in which the endosperm could be of the usual vitreous type.

## **5. Increased synthesis of other proteins**

An important consequence of silencing of the prolamine genes in cereals is an increase in the synthesis of other proteins, including those with a higher content of essential amino acids. For example, in transgenic maize plants with α-zein silencing, a double content of tryptophan and lysine was observed [40]. In rice, it was shown that silencing of 13 kDa prolamine increases the total lysine content up to 56% as a result of a compensatory increase in the synthesis of lysine-rich glutelin, globulins, and chaperones [41]. A significant increase in the lysine content (up to 3.3 g / 100 g of protein, compared to 2.1 g/100 g of protein in the non-transgenic control) was found in transgenic sorghum plants carrying complex genetic constructs for RNA silencing of kafirins (ABS032, ABS149) [35]. However, these genetic constructs carried, along with the fragments of the kafirin genes, the fragments of the lysine ketoglutarate reductase gene, which controls the catabolism of free lysine. This fact does not allow drawing a conclusion on the effect of kafirin silencing on the increase in the lysine content in sorghum.

*Grain and Seed Proteins Functionality*

formation of floury endosperm [38].

RNAi mutant #1–1 of cv. Avans, had also a floury type of endosperm (**Figure 4**). It should be noted that, in similar experiments in maize, silencing of different zein genes also resulted in reduction of the vitreous endosperm and formation of kernels with floury endosperm [37–39]. It was shown that γ-zein gene plays an important role in the formation of the floury endosperm, and silencing of this gene modified the structure of protein bodies and their connection with starch grains that result in

Unfortunately, the presence of floury endosperm is a significant disadvantage of the obtained lines, since the absence of a vitreous layer increases the fragility of the kernels and reduces its resistance to fungal diseases. It should be noted that the floury (opaque) type of endosperm is characteristic of the P721Q mutant and many

*Longitudinal sections of kernels of the original non-transgenic line Zheltozernoe-10 (A) and transgenic plants carrying pNRKAF genetic construct for RNA silencing of the γ-KAFIRIN-1 gene (B-F), differing in the degree*

*of development of the vitreous endosperm. Vitreous endosperm is marked with white arrows.*

**150**

**Figure 5.**

In the transgenic plants obtained in our experiments with a high *in vitro* kafirin digestibility, the total amino acid content in the kernels of plants of the T2 generation decreased by 22.8–40.2% as compared with the original, non-transgenic line [23]. At the same time, the relative content of the two main essential amino acids, lysine and threonine, has increased significantly. The proportion of lysine increased 1.6–1.7 times: from 1.54% of the total amino acid content in the flour of the original non-transgenic line to 2.41–2.63% in transgenic plants. This increase, combined with a significant decrease in the total level of amino acids, was apparently caused by a decrease in the content of α-kafirins, which are poor in lysine and threonine, while the synthesis of other proteins was not impaired. Accordingly, the relative proportions of lysine and threonine increased. It is possible that suppression of the synthesis of γ-kafirin prevents the accumulation of α-kafirins but does not affect the synthesis of other proteins richer in lysine and threonine. The appearance of new proteins in transgenic sorghum plants carrying a genetic construct for silencing α-kafirin gene was described by T. Kumar et al. [21].

It is noteworthy that in the transgenic plants of the cv. Avans with a construct for silencing γ-*KAFIRIN-1* (RNAi mutant #1–1), along with a decrease in the content of γ- and α-kafirins (**Figure 1**), an increase in the content of a number of globulins occurs, possibly resulting from the re-balancing of the proteome of the kernels (**Figure 6**).

Protein rebalancing in the endosperm is a frequent phenomenon in transgenic plants with genetic constructs for RNA silencing of seed storage proteins. In maize, it was suggested that a compensatory mechanism, which is sensitive to the protein

#### **Figure 6.**

*Electrophoretic spectra of globulins from the kernels of transgenic plants from T1 generation of RNAi mutant #1–1. 1 – Original non-transgenic cv. Avans; 2–7 individual T1 plants; M – Molecular mass markers (kDa). Globulins were extracted according to [42].*

**153**

**Figure 7.**

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage…*

content exists in the kernels; and a violation of zein synthesis in developing kernels enhances the translation of other mRNAs [43]. It is noteworthy that in transgenic soybean plants with suppressed synthesis of the main storage proteins, the seeds retained an almost identical level of total protein characteristic of untransformed soybean varieties [44]. These data suggest that restoration of proteome balance may be quite common phenomenon, providing a constant supply of nitrogen during

In our experiments, we found that the offspring of transgenic plants with a high *in vitro* digestibility of endosperm proteins sometimes lose this trait. Even different panicles of the same plant had different digestibility values. Such instability is an interesting phenomenon, which may be caused by silencing of introduced genetic construct possibly by RNA-dependent DNA methylation that is characteristic to hairpin genetic constructs [45], or by environmental factors, such as temperature, soil moisture, air humidity, etc. It has been reported that temperature causes a significant impact on RNAi-silencing [46]. It was also shown that mRNA degradation induced by microRNA and translation inhibition, depends on the temperature of plant growth [47]. Consequently, the efficiency of inhibition of kafirin synthesis by RNAi-silencing may be sensitive to plant growing conditions, and this was really

*PCR analysis of plants from the offspring of the RNAi mutant (#1–1, cv. Avans) carrying the genetic construct for silencing γ-KAFIRIN-1, with primers to the nos-promoter (A) and ubi1-intron (B). 1 – Original non-transgenic cv. Avans; 2-4 (A) and 2-5 (B) – individual T1 plants (A: #2, #3, #4; B: #1, #2, #3, #4, respectively); 5–14(A) and 6–15 (B) – Plants from another experiment; 15 (A), 16 (B) – A. tumefaciens GV3101/pNRKAF; 16 (A), 17 (B) – DNA markers; 17 (A), 18 (B) – Negative control (no DNA). The nosspecific primers amplified the 202 bp fragment (A). The ubi1-intron specific primers amplified the 588 bp*

*fragment (B). The arrows mark the products of DNA amplification in plant #3.*

**6. Instability of the genetic construct for RNA silencing**

*DOI: http://dx.doi.org/10.5772/intechopen.96204*

seed maturation.

shown in our experiments [48].

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage… DOI: http://dx.doi.org/10.5772/intechopen.96204*

content exists in the kernels; and a violation of zein synthesis in developing kernels enhances the translation of other mRNAs [43]. It is noteworthy that in transgenic soybean plants with suppressed synthesis of the main storage proteins, the seeds retained an almost identical level of total protein characteristic of untransformed soybean varieties [44]. These data suggest that restoration of proteome balance may be quite common phenomenon, providing a constant supply of nitrogen during seed maturation.

### **6. Instability of the genetic construct for RNA silencing**

In our experiments, we found that the offspring of transgenic plants with a high *in vitro* digestibility of endosperm proteins sometimes lose this trait. Even different panicles of the same plant had different digestibility values. Such instability is an interesting phenomenon, which may be caused by silencing of introduced genetic construct possibly by RNA-dependent DNA methylation that is characteristic to hairpin genetic constructs [45], or by environmental factors, such as temperature, soil moisture, air humidity, etc. It has been reported that temperature causes a significant impact on RNAi-silencing [46]. It was also shown that mRNA degradation induced by microRNA and translation inhibition, depends on the temperature of plant growth [47]. Consequently, the efficiency of inhibition of kafirin synthesis by RNAi-silencing may be sensitive to plant growing conditions, and this was really shown in our experiments [48].

#### **Figure 7.**

*Grain and Seed Proteins Functionality*

(**Figure 6**).

α-kafirin gene was described by T. Kumar et al. [21].

In the transgenic plants obtained in our experiments with a high *in vitro* kafirin digestibility, the total amino acid content in the kernels of plants of the T2 generation decreased by 22.8–40.2% as compared with the original, non-transgenic line [23]. At the same time, the relative content of the two main essential amino acids, lysine and threonine, has increased significantly. The proportion of lysine increased 1.6–1.7 times: from 1.54% of the total amino acid content in the flour of the original non-transgenic line to 2.41–2.63% in transgenic plants. This increase, combined with a significant decrease in the total level of amino acids, was apparently caused by a decrease in the content of α-kafirins, which are poor in lysine and threonine, while the synthesis of other proteins was not impaired. Accordingly, the relative proportions of lysine and threonine increased. It is possible that suppression of the synthesis of γ-kafirin prevents the accumulation of α-kafirins but does not affect the synthesis of other proteins richer in lysine and threonine. The appearance of new proteins in transgenic sorghum plants carrying a genetic construct for silencing

It is noteworthy that in the transgenic plants of the cv. Avans with a construct for silencing γ-*KAFIRIN-1* (RNAi mutant #1–1), along with a decrease in the content of γ- and α-kafirins (**Figure 1**), an increase in the content of a number of globulins occurs, possibly resulting from the re-balancing of the proteome of the kernels

Protein rebalancing in the endosperm is a frequent phenomenon in transgenic plants with genetic constructs for RNA silencing of seed storage proteins. In maize, it was suggested that a compensatory mechanism, which is sensitive to the protein

*Electrophoretic spectra of globulins from the kernels of transgenic plants from T1 generation of RNAi mutant #1–1. 1 – Original non-transgenic cv. Avans; 2–7 individual T1 plants; M – Molecular mass markers (kDa).*

**152**

**Figure 6.**

*Globulins were extracted according to [42].*

*PCR analysis of plants from the offspring of the RNAi mutant (#1–1, cv. Avans) carrying the genetic construct for silencing γ-KAFIRIN-1, with primers to the nos-promoter (A) and ubi1-intron (B). 1 – Original non-transgenic cv. Avans; 2-4 (A) and 2-5 (B) – individual T1 plants (A: #2, #3, #4; B: #1, #2, #3, #4, respectively); 5–14(A) and 6–15 (B) – Plants from another experiment; 15 (A), 16 (B) – A. tumefaciens GV3101/pNRKAF; 16 (A), 17 (B) – DNA markers; 17 (A), 18 (B) – Negative control (no DNA). The nosspecific primers amplified the 202 bp fragment (A). The ubi1-intron specific primers amplified the 588 bp fragment (B). The arrows mark the products of DNA amplification in plant #3.*

#### **Figure 8.**

*PCR analysis of transgenic sorghum plants (T4 generation) carrying a genetic construct pNRKAF [23] with primers to the ubi1-intron (A) and nos-promoter (B). 1 (A, B) – Original non-transgenic line Zh10; 2–-14 (A), 2-12 (B) – DNA of individual transgenic plants from the T4 families; 15 (A), 14 (B) – A. tumefaciens GV3101/pNRKAF (positive control); 16 (A), 14 (B) – DNA markers; 15 (A) – Negative control (no DNA). The ubi1-intron specific primers amplified the 267 bp fragment (A). The nos-specific primers amplified the 202 bp fragment (B). Amplified gene-specific fragments are marked with arrows [50].*

In addition to instability at the epigenetic level, we have found the genetic instability of introduced construct for RNAi-silencing. In this regard, analysis of the progeny of the RNAi mutant #1–1 (cv. Avans), carrying a construct for silencing γ-*KAFIRIN-1*, is indicative. Of the 4 studied T1 plants grown in the experimental field plot, all plants were transgenic, because carried the *nos*-promoter driving the expression of the marker gene *bar*, located in T-DNA of pNRKAF, along with a genetic construct for the γ-*KAFIRIN-1* gene silencing (**Figure 7A**). At the same time, one of these plants (#3) lacked the *ubi1*-intron, which is a part of the genetic construct for silencing (**Figure 7B**). All kernels developed in the panicle of plant #3 had the vitreous type of endosperm, characteristic to the original cultivar (**Figure 4A**), while in the panicles of other plants, in which the *ubi1-*intron was present, the kernels had a floury type of endosperm (**Figure 4B**), characteristic for transgenic plants with γ-kafirin silencing.

In addition, in Zh10 transgenic plants from the T4 families with high digestibility of kafirins, probable elimination of the *nos*-promoter, which controls the expression of the marker gene *bar* in the pNRKAF genetic construct [23] was found [49]. **Figure 8** clearly shows that in the plants from the T4 families, amplification of the *ubi1-*intron fragment was observed, while amplification of the *nos-*promoter located in the construct in front of the marker gene *bar* was absent. Thus, these plants probably turned out to be functionally marker-free transgenic plants. This fact is of significant interest, since the presence of marker genes in the genetic constructs hinders the practical use of transgenic lines in practical plant breeding.

**155**

**Author details**

**Acknowledgements**

grant 19-016-00117.

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage…*

The research findings presented in this chapter provide strong evidence that RNA interference can be used for the improvement of the nutritional value of grain sorghum. RNAi mutants are characterized by significantly improved digestibility of kafirins and higher content of essential amino acids, in particular lysine. In some cases, these mutants retain vitreous endosperm that is highly important for grain

Nevertheless, in most cases the kernels with suppressed synthesis of γ- or α-kafirins have floury endosperm that strongly reduces their use in sorghum breeding. Such a correlation between the traits of high digestibility of kafirins and the floury type of endosperm, which was originally observed in the P721Q mutant and lines created on its basis is a serious problem (see review [8]). In maize, the correlation between the floury endosperm and the increased lysine content was disrupted using modifier genes that enhanced the accumulation of γ-zein [42, 51, 52]. However, in sorghum, an increase in the synthesis of γ-kafirin may decrease the level of kafirin digestibility due to a high content of sulfurcontaining amino acids, which contribute to the polymerization of kafirins. Possibly, one of the ways to solve this problem may be down-regulation of genes that encode protease inhibitors, which can also affect the level of digestion of kafirins by exogenous proteases. In this case, the resulting lines would have a hard

hardiness and in ensuring the resistance of kernels to fungal diseases.

endosperm in combination with a high digestibility of kafirins.

Lev A. Elkonin\*, Valery M. Panin, Odissey A. Kenzhegulov and Saule Kh. Sarsenova

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

The work was funded in part by the Russian Foundation for Basic Research,

Federal Agricultural Research Centre of South-East, Saratov, Russia

\*Address all correspondence to<EMAIL_ADDRESS>
provided the original work is properly cited.

*DOI: http://dx.doi.org/10.5772/intechopen.96204*

**7. Conclusions**

*RNAi-Mutants of* Sorghum bicolor *(L.) Moench with Improved Digestibility of Seed Storage… DOI: http://dx.doi.org/10.5772/intechopen.96204*


**Meet the editor**

Dr. Gemma Aiello was born in Aversa (CE), Italy, on 24 October 1964. In 1989, she graduated in Geological Sciences at the University of Naples "Federico II." In 1993, she earned a PhD degree in Sedimentary Geology at the University of Naples "Federico II," Department of Earth Sciences, Faculty of Geological Sciences. She completed a 2-year postdoctoral fellowship at the University

of Naples "Federico II," a CNR-CEE fellowship, and several contracts at the Research Institute "Geomare Sud," CNR, Naples, Italy. Since 1998, she has been a full-time researcher at the Italian CNR. Dr. Aiello has a 25 year experience in the field of sedimentary geology, marine geology, and geophysics, participating in different research projects of Italian National Research Council (CARG, Vector, Centri Regionali di Competenza). She was a contract professor of sedimentology and stratigraphy at the Parthenope University of Naples, Italy, and a teacher in formation courses of technicians in marine science and engineering in Naples, Italy.


**Author details**

*Acoustics of Materials*

<EMAIL_ADDRESS>
at<EMAIL_ADDRESS>
**Biographies and contact information quantum physics**

College, and he can be reached at<EMAIL_ADDRESS>
Ayax D. Ramirez joined SPAWAR Systems Center, Diego in 1997. He received his M.S. and B.S. in Physics from San Diego State University in 1991 and 1984, respectively. Since 1984, he has performed research and development in the areas of laser physics, photonics, micro-sensors, excimer laser materials processing, and, more recently, in nondestructive detection techniques in high-power microwave tubes. Prior to working at SSC-SD, he worked in industry as a researcher for Science Applications International Corporation (SAIC). He holds numerous patents and technical publications in his field. He is a member of the American Physical Society and an adjunct professor at Southwestern College and Grossmont

Stephen D. Russell, Head of the Advanced Technology Branch at SPAWAR Systems Center, received his Ph.D. and M.S. in Physics from The University of Michigan in 1986 and 1981, respectively. He received his B.S. in Physics and Earth & Space Science from the State University of New York at Stony Brook in 1979. He has worked at SPAWAR Systems Center (and its predecessor organizations) since 1986 performing research and development in the fields of micro-sensors, photonic devices and systems, microelectronics, excimer laser processing, novel materials, and display technologies. Prior to joining SPAWAR Systems Center, he worked as a research assistant at The University of Michigan (1979–1986) and at Brookhaven National Laboratory (1977–1978). He has published numerous technical articles and has more than 60 U.S. and foreign patents issued or pending. He is a member of the American Physical Society and Optical Society of America and also serves as a consultant and technology agent for the Missile Defense Agency (MDA) and Defense Advanced Research Projects Agency (DARPA). He is a 2001 recipient of the Federal Laboratory Consortium Award for Excellence in Technology Transfer and holds two NASA Space Act Awards. He can be reached at

David W. Brock, a Scientist in the Antennas and RF Technology Branch at SPAWAR Systems Center, received his Ph.D. in Physics from the University of Texas at Austin in 1981 and his M.S. in Physics from the University of Colorado at Boulder in 1975. He received B.S. degree in Physics and Mathematics at the New Mexico Institute of Mining and Technology in 1972. Prior to joining SPAWAR Systems Center, Dr. Brock worked as a research assistant at the University of Texas (1977– 1981) and the University of Colorado (1975). He has worked at SPAWAR Systems Center since 1981 initially in the High Power Microwave Tube Branch and later in the Microwave/Millimeter Wave Technology Branch. He has carried out research work in areas including high power microwave tubes, the design and building of radar systems, microwave receivers, and antennas. He has published several technical articles, and has seventeen patents issued or pending. Dr. Brock can be reached

Narayan R. Joshi, faculty at the Welding Technology Department of North Harris College, received his M.Sc. in Electronics from Poona University, India. He received his M.S. and Ph.D. in Mechanics and Materials Science from Johns Hopkins University. He has worked as the Head of the dual Engineering Degree at the University of Maryland, Baltimore; Lamar University; and Prairie View A&M University. His academic teachings include elastic wave propagation, Materials Science, Metallurgy, Mechanical Vibrations, and Advanced Non-Destructive Evaluation Methods. His experimental research includes Ultrasonics, Acoustic Emission, X-ray diffraction and Optical Materials (Lasers without Inversion). Mr. Joshi has worked for General Electric, Oak Ridge National Laboratories, Southwest Research Institute, and Texas Instruments. He has about 55 research

**56**

Ayax D. Ramirez1 \*, Stephen D. Russell1 , David W. Brock1 and Narayan R. Joshi2

1 Space and Naval Warfare Systems Center, Pacific, San Diego, CA, USA

2 North Harris College, Houston, TX, USA

\*Address all correspondence to<EMAIL_ADDRESS>
© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.