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UDC 602.62:581.143.5:634.675:582.926 https://doi.org/10.15407/biotech12.02.056
DIRECT PLANT REGENERATION FROM Pysalis peruviana L. EXPLANTS
O. M. Yaroshko Institute of Cell Biology and Genetic Engineering
M. V. Kuchuk of the National Academy of Sciences of Ukraine, Kyiv
E-mail: 90tigeryaroshko90@gmail.com
Received 25.02.2019 Revised 28.03.2019 Accepted 10.05.2019
The aim of the work was to establish the effective culture medium for the regeneration of Physalis peruviana for further micropropagation and obtaining of adult plants from regenerants in vitro conditions. After conducting series of experiments, effective culture media for the regeneration of Ph. peruviana was established. The most effective media for shoot regeneration from leaf explants were MS30 supplemented with 1mg/l Kin + 3 mg/l BAP; MS30 + 2 mg/l Kin + 1 mg/l BAP (33.33% of regeneration on both media). Good results were obtained on the media MS30 supplemented with 1 mg/l Kin and 2 mg/l BAP (28.57% explants regenerated) and MS30 supplemented with 2 mg/l Kin and 3 mg/l BAP (26.31% of regeneration). Root induction from stem and leaf explants were obtained on medium MS30 with NAA (0.2 mg/l; 0.5 mg/l), IAA (0.2 mg/l; 0.5 mg/l). Root induction frequency on these media was 100%. The obtained regenerants were separated from the explants and were transferred on the medium MS30 with 1 mg/l of BAP for elongation, and then on a medium MS30 or MS30 with 0.2 mg/l NAA for subsequent rooting. After one month of cultivation on mediums MS30 or MS30 with 0.2 mg/l NAA were successfully received adult plants.
Key words: Physalis, regeneration.
Due to the medicinal and horticultural values Physalis peruviana is widely cultivated in tropical and subtropical countries. Physalis finds its application in medicine due to rich biochemical composition (the main components are 15-desacetylphysabubenolide and betuline). Physalis species have antitumor effect and used to treat inflammations.
Ph. peruviana is highly productive plant. From one plant it is possible to collect about 300 fruits.
In Ukraine, Ph. peruviana is grown only in private collections. It is not grown on an industrial scale, therefore, in the case of obtaining transgenic Physalis plants, it is easier to prevent the possible leakage of unauthorized transgenes.
According to mentioned above information, Physalis is a promising plant for the production of recombinant proteins for pharmaceutical use.
A sensational article about editing of Ph. pruinosa genome was recently published [1]. Physalis can be a good model object for studying the functioning of heterologous genes in its tissues and organs.
At present, there are many works devoted to the callus formation and regeneration of Physalis. Basically, researchers who obtained regenerants had the main goal of using them as a source of secondary metabolites and other valuable substances, therefore the largest number of works devoted to the study of Physalis has a biochemical direction.
The study of regenerative ability was undertaken by a group headed by Rao. They obtained regenerants for Ph. pubescence. Initially, they received a callus tissue from the leaves and internodes. Then, regenerants were received on the medium MS30 + 2 mg/l BAP + 0.5 mg/l NAA and on the medium MS30 + 2.5 mg/l BAP + 0.5 mg/l NAA, from the callus tissue [2].
Ramar K. and Ayyadurai V. chose Physalis maxima as an object of research. They managed to obtain regenerants for this specie from the leaf segments on the media: MS30 + 1 mg/l BAP + 0.5 mg/l NAA; MS30 + 2 mg/l BAP + 1 mg/l NAA + 1 mg/l Kin and regenerants from nodal segments on mediums: MS30 + 2 mg/l BAP + 1.5 mg/l NAA + 0.5 mg/l
GA3; MS30 + 3 mg/l BAP + 1.5 mg/l; NAA + 1.5 mg/l GA3 [3].
Sandhya H., and Srinath R. received regenerants from nodal segments of Physalis minima on media: MS30 + 2 mg/l 2.4 — D + 2 mg/l NAA; MS30 + 2 mg/l 2.4 — D + 1 mg/l Kin [4].
Ramar K. with colleagues received a positive regeneration result for Ph. peruviana on media: MS30 + 1.5mg/l BAP + 0,5 mg/l GA3 + 0.5 mg/l 2.4 — D; MS30 + 2 mg/l BAP + 1mg/l GA3 + 1 mg/l 2.4 — D (for nodal and internodal segments); and on media MS30 + 2.5 mg/l BAP + 1 mg/l GA3 + 0.5 mg/l 2.4 — D; MS30 + 3 mg/l BAP + 1 mg/l GA3 + 1 mg/l 2.4 — D (for leaf explants) [5].
Bergier K. and colleagues received Ph. ixocorpa regenerants from the hairy root's culture on MS30 + 5 pM Kin + 1 pM BAP [6].
Kumar O.A. and colleagues received regenerants of Ph. angulata from meristems on medium MS30 + 1 mg/l BAP + 0.5 mg/l IAA + 0.25 mg/l GA3 [7].
Swartwood K. and Van Eck obtained Ph. pruinosa regenerants from hypocotyls on medium MS30 + 2 mg/l ZEA [8].
Assad-Garcia N. received regenerants from the cotyledons of 12-day's seedlings of Ph. ixocorpa cv. Rendidora on medium MS30 + 1 pM NAA + 12.5 pM BAP [9].
Singh P. and colleagues received regenerants from the nodal segments of Ph. peruviana on medium MS30 + 2.5 mg/l BAP + 0.05 mg/l IBA [10].
Several scientific groups worked with Ph. minima. Regenerants of this species were obtained from apical meristems of 15-day — old seedlings and nodal segments [11]; from the callus on the medium MS30 + 1 mg/l BAP + 1 mg/l Kin + 3.5 mg/l GA3 [12]; from nodal segments on medium MS30 + 2 mg/l BAP + 0.25 mg/l IAA [13]; from callus on medium MS30 + 3.5 mg/l BAP + 0.4 mg/l Kin [14]; from the apical meristems of seedlings and nodal segments on medium MS30 + 1 mg/l BAP [15].
Although a sufficient quantity of works dedicated to the regeneration of Ph. peruviana, an effective protocol has not yet been developed for obtaining a large number of regenerants from Physalis leaf explants.
Our objective was to establish effective culture medium for the regeneration of Ph. peruviana for further obtaining of adult plants from regenerants in vitro conditions.
Materials and Methods
The objects of the research were Ph. peruviana plants.
Seeds of Physalis germinated on the sterile nutrient agar medium Murashige and Skoog (MS30) [16] with 30 g/l sucrose (22-26 °C, 14-hour light period, illumination — 3000-4500 lx).
For regeneration we used the internodes' segments, segments of leaflets, petioles of leaflets and leaflets with petioles (without separation) 1 cm long, derived from 1-month-old plants of Ph. peruviana. The explants were cultivated horizontally for 1 month on MS30 medium, containing 30 g/l sucrose (pH 5.75.9) with the addition of 6-benzylaminopurine (BA), 1-naphthylacetic acid (NAA), kinetine (Kin) in different concentrations.
Obtained shortened shoots were separated and transferred for 2 weeks on MS30 medium with 1 mg/l of BAP for elongation, and then on a medium MS30 or MS30with 1 mg/l NAA for subsequent rooting.
Data collection and Statistical analysis
For each experiment, 15 explants were used. Data was analyzed using the general procedure of Statistica software package, Version 12. When the P indicated significant treatment effects (5, 1 or 0,1%) based on the Spearman's rank order correlation (R), the Least Significant Difference test (P < 0,05; P <0,01; P < 0,001) was used as a method to determine which treatments were significantly different from others treatments. The significance levels (P) of averages differences or relations of the mean values were determined by tables for small samples.
We carried out Spearman analysis. In order to confirm the validity of the results, the comparison of the group values was carried out. The comparison with control wasn't carried out, as for the control group we have value "0".
The control group were plants, parts of which were placed on the medium without the addition of stimulants. experimental groups — plants placed on medium with growth stimulants. No changes were observed in the control group (therefore, in the control group 0).
The Spearman's rank correlation coefficient is used to identify and assess the closeness of the connection between two ranks of compared quantitative indicators.
The correlation coefficient can take values from -1 to 1, and with R = 1 there is
a strictly direct connection, and with R = -1 there is a inverse connection. If the correlation coeffici e nt is zero, then the relationship between t he values is practically absent. The closer the correlation coefficient to one, the stronger is the connection between the measured values.
When usi n g the rank correlation coefficient, conditionally assess the closeness of the c o nnection between the signs, considering the values of the coefficient equal to 0.3 or less — indicators of weak closeness of the connection; values of more than 0.4, but less than 0.7 are indicators of moderate closeness of connection, and values of 0.7 or more are indicators of high closeness of connection.
In our work we compared the connection of effect of growth regulators (which was expresse d in the appearance of different quantity of regenerants per one explant) with used concentrations & combinations of growth regulators.
Results and Discussion
After cultivation explants on MS30 medium with different concentrations of BAP and Kin were obtained explants (Fig. 1, 2).
Highest levels of regeneration were obtained, while cultivating the leaf explants on media MS30 with 2 mg/l Kin + 3 mg/l BAP and MS30 with 2 mg/l Kin + 1 mg/l BAP (Fig. 1). Quite good levels of regeneration were received on media MS30 with 1 mg/l Kin + 2 mg/l BAP and MS30 2 mg/l Kin + 3 mg/l BAP. Low levels of regeneration were received on medium MS30 with 1 mg/l Kin + 1 mg/l BAP; 1 mg/l Kin + 2 mg/l BAP; 2 mg/l BAP; 2 mg/l BAP + 2 mg/l Kin; 3 mg/l BAP; 4 mg/l BAP + 1 mg/l Kin; 4 mg/l BAP + 2 mg/l Kin. The explants
cultured on MS30 medium without addition of growth regulators, didn't regenerate (Table 1, 2; Fig. 2). Also, there weren't obtained regeneration on medium with addition only cytokinins (MS30 + 1-2 mg/l Kin). Absence of regeneration was also on medium with addition only low or high amounts of auxins (MS30 + 1 mg/l BAP, MS30+ 4 mg/l BAP) (Table 1).
According to our result, the most effective media for shoot regeneration were MS30 + 1 mg/l Kin + 3 mg/l BAP and MS30 + 2 mg/l Kin + 1 mg/l BAP (33,33% of regeneration on both mediums) (Fig. 2, 3). Also, quite good results were obtained on the media MS30 + 1 mg/l Kin + 2 mg/l BAP (28.57% explants regenerated) and MS30 + 2 mg/l Kin + 3 mg/l BAP (26.31% of regeneration) (Fig. 2, 3).
Also sev e ral research groups obtained positive results for regeneration of species of Physalis genus. In majority of works were used growth regulators BAP and Kin with addition of 3-rd growth regulator [3, 5, 7, 12]. We decide to simplify the methodic of regeneration and use o nly 2 growth regulators: BAP (concentration 0-4 mg/l) with Kin (0-2 mg/l). According previous works, highest frequency of regen e ration was obtained on mediums, which contain BAP (concentration 1-3 mg/l), Kin 1 mg/l [3, 6, 12]. In our results, highest frequency of regeneration was obtained while using th e same concentrations of BAP and Kin. It w as claimed, that mean quantity of regenerated plants was about 11-13 pieces per one explants which is matches our results (for several variants of mediums, mean quantity of regenerants per one explant was 11-12 pc.).
For root induction was used MS30 medium with NAA (0.2 mg/l; 0.5 mg/l), IAA (0.2 mg/l; 0.5 mg/l). No significant difference was found between t he media used for root induction (Fig. 4).
A
B
Fig. 1. Shoot induction from leaf explants in MS30 medium with plant growth regulators (A — 2 mg/l Kin + 3 mg/l BAP; B — 0 mg/l Kin + 3 mg/l BAP) after 1 month of cultivation
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Fig. 2. Effect of growth regulators on frequency of shoot regeneration from leaf segments of Physalis peruviana in MS30 medium after 1 month of cultivation
Data are the mean ±SE (standard error); n = 15
Table 1. Effect of growth regulators on shoot induction of Physalis peruviana from leaf explants on MS30 medium
0 mg/l BAP 1 mg/l BAP 2 mg/l BAP 3 mg/l BAP 4 mg/l BAP
0 mg/l Kin - - * * -
1 mg/l Kin - * ** *** *
2 mg/l Kin - *** * ** *
Where - no regeneration;
* — frequency of regeneration from14% to 20%; ** — frequency of regeneration from 20% to 30%; *** —frequency of regeneration more than 30%.
Table 2. Effect of growth regulators on quantity of regenerated shoots of Physalis peruviana from leaf explants on MS30 medium
0 mg/l BAP 1 mg/l BAP 2 mg/l BAP 3 mg/l BAP 4 mg/l BAP
0 mg/l Kin - - 6 ± 1.15 6 ± 1.15 -
1 mg/l Kin - 10 ± 0.84 11.8 ± 0.882 12.5 ± 0.72 11.2 ± 0.805
2 mg/l Kin - 12.3 ± 0.76 11.6 ± 0.93 12 ± 0.85 9 ± 0.654
Quantity of shoots (M±SE).
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Fig. 3. Effect of growth regulators on mean number of shoot of Physalis peruviana in MS30 medium after 1 month of cultivation
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Fig. 4. Root induction from leaf explants in MS30 medium supplemented with various plant growth regulators after one month of cultivation
Obtained shortened shoots were separated and transferred for 2 weeks on MS30 medium with 1 mg/l of BAP for elongation, and then on a medium MS30 or MS30 with 0,2 mg/l NAA for subsequent rooting (Fig. 5).
Statistical analysis
After summarizing obtained results, statistical analysis was conducted for data. The results of regeneration (number of shoots per explants) were significantly different from those which were obtained without treatment with growth regulators and when comparing the effect of different growth regulators.
Null hypothesis is rejected, with significant (P < 0,05); highly significant (P < 0,01) and extremely significant (P < 0,001) levels of averages differences.
Fig. 5. Rooted young regenerants on MS30 medium
According Spearman's rank correlation, there we r e direct moderate closeness of connecti o n between used concentration of growt h regulators and quantity of regenerants per 1 expiant (in most cases). Also were found inverse connection between used concentration of growth regulators and quantity of regenerants per 1 explant, when the concentration of BAP growth regulator was 4 mg/l. Thus, it can be argued that the concentration of 4 mg/l BAP regulator had a depressing effect on regeneration.
Thus, ef f ective culture media for the regenera t ion of Physalis peruviana were established. The most effective media for shoot regeneration from leaf explants were MS30 + 1 mg/l Kin + 3 mg/l BAP and MS30 + 2 mg/l
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ПРЯМА РЕГЕНЕРАЦ1Я РОСЛИН Physalis peruviana L. З ЕКСПЛАНТ1В
О. М. Ярошко, М. В. Кучук
1нститут клггинно! бмлогп та генетично! шженерп НАН Укра!ни, Ки!в
E-mail: 90tigeryaroshko90@gmail.com
Метою роботи було виявлення ефективно-го культурального середовища для регенерацп Physalis peruviana з метою його подальшого розмноження i отримання дорослих рослин з регенеранмв за умов in vitro. Шсля проведен-ня серп експерименив було пЩбрано ефек-тивш живильш середовища для регенерацп P.peruviana. Найбшьш ефективними середови-щами для регенерацп пагошв з листових екс-планив були МС30, доповнене 1 мг/л Кш та 3 мг/л БА i МС30, доповнене 2 мг/л Kiн та 1 мг/л БА (33,33% регенерацп на обох середовищах). Хорошi результати було отримано на середовищах МС30 з додаванням 1 мг/л Юн та 2 мг/л БА (28,57% експлантамв регенерували) i МС30 з 2 мг/л Юн та 3 мг/л БА (ефектившсть регенерацп — 26,31%). 1ндукщю корешв на стебло-вих i листових експлантатах було отримано на середовишД МС30 з додаванням НОК (0,2 мг/л; 0,5 мг/л), 1ОК (0,2 мг/л; 0,5 мг/л). Частота ко-ренеутворення на цих середовищах становила 100%. Одержат регенеранти вщокремлюва-ли вщ експланмв i переносили на середовище МС30 або МС30 з 0,2 мг/л НОК для подальшого вкоршювання. Пiсля одного мiсяця культиву-вання на середовищах МС30 або МС30 з 0,2 мг/л НОК було отримано доро^ рослини.
Ключовi слова: Physalis, регенеращя.
ПРЯМАЯ РЕГЕНЕРАЦИЯ РАСТЕНИЙ Physalis peruviana L. ИЗ ЭКСПЛАНТОВ
О. Н. Ярошко, Н. В. Кучук
Институт клеточной биологии и генетической инженерии НАН Украины, Киев
E-mail: 90tigeryaroshko90@gmail.com
Целью работы было выявление эффективной культуральной среды для регенерации Physalis peruviana с целью его дальнейшего размножения и получения взрослых растений из регенерантов в условиях in vitro. После проведения серии экспериментов были подобраны эффективные питательные среды для регенерации P. peruviana. Наиболее эффективными средами для регенерации побегов из листовых эксплантов были МС30, дополненное 1 мг/л Кин и 3 мг/л БА, и МС30, дополненное 2 мг/л Кин и 1 мг/л БА (33,33% регенерации на обеих средах). Хорошие результаты были получены на средах МС30 с добавлением 1 мг/л Кин и 2 мг/л БА (28,57% эксплантатов регенерировали) и МС30 с 2 мг/л Кин и 3 мг/л БА (эффективность регенерации составила 26,31%). Индукция корней на стеблевых и листовых эксплантатах получена на среде МС30 с добавлением НУК (0,2 мг/л; 0,5 мг/л), ИУК (0,2 мг/л; 0,5 мг/л). Частота корнеобразования на этих средах составила 100%. Полученные регенеранты отделяли от эксплантов и переносили на среду МС30 или МС30 с 0,2 мг/л НУК для последующего укоренения. После одного месяца культивирования на средах МС30 или МС30 с 0,2 мг/л НУК были получены взрослые растения.
Ключевые слова: Physalis, регенерация.
