3.

Plant growth regulators optimization for in vitro cultivation of the orchid

Guarianthe skinneri (Bateman) Dressier & W.E.Higgins. Christian Yanelly
Coello1 , Clara Luz Miceli1 , Carolina Orantes1 , Luc Dendooven2 &
Federico Antonio Gutiérrez. Gayana Bot. 67(1): 19-26, 2010

Today I want to tell you some words about the article I`ve read recently. It
describes the optimization of the plant growth in vitro regulators for one of the
orchid species called Guariathe skinneri. In 2010, a group of botanical scientists,
Christian Yanelly Coello, Clara Luz Miceli, Carolina Orantes, Luc Dendooven
and Federico Antonio Gutiérrez published the results of research in Gayana Bot.
The main aim was to standardize the protocol for mass propagation and ex-situ
conservation of this specie.
[ For the research, 5 capsules of Guariathe skinneri were taken. They were
washed and sterilized with ethanol. Next, 2 seeds were placed in each of the 150
sterilized tubes with MS basal medium with the addition of sucrose. Incubated
under cold fluorescent light in a photoperiod of 16/8 light/dark at 25-27 degrees.
After 2 weeks, the seedlings were moved to larger bottles. Different
concentrations of the hormones GA3, NAA, BA, and IAA were added to these
bottles . After that, the seedlings with shoots and roots were transferred to pots
with a mixture of peat moss and agrolite for hardening in ambient light
conditions. After 4 weeks, the seedlings were transferred to the greenhouse. The
Statistica (2000) software was used for the analysis. The analysis showed that the
most important factor in the growth of shoots and the number of roots is
gibberellic acid (GA3). The physiological effect of GA3 on plant growth is well
known, as it increases the concentration of soluble carbohydrates during seed
germination and plant growth. ]
It was found that GA3 was the most important factor for shoot and root
induction and elongation. However, the optimal concentrations were small and
larger concentrations had a negative effect on plant development.
For me, this article, as well as the given research data, were sufficiently
informative. The results of this study present new evidence regarding the effect
of GA3 to micropropagate G. skinneri through protocorms and this protocol could
be useful for other orchids. As for me, I`m making the similar research for my
diploma thesis and have found a lot of points, that might be useful in this article.
Для исследования было взято 5 капсул Guariathe skinneri. Их промыли и
стерилизовали этанолом. Далее в каждую из 150 стерилизованных
пробирок с питательной средой MS с добавлением сахарозы поместили по
2 семени. Инкубировали при холодном флуоресцентном свете и
фотопериоде 16/8 свет/темнота при 25-27 градусах. Через 2 недели всходы
переместили в бутылки большей емкости. В эти бутылки добавили разные
концентрации гормонов GA3, NAA, BA и IAA . После проростки с побегами
и корнями перенесли в горшки со смесью торфяного мха и агролита для
закаливания в условиях рассеянного света. Спустя 4 недели саженцы
перенесли в теплицу. Для анализа использовали программу Statistica (2000).
Анализ показал, что наиболее важным фактором роста побегов и
количества корней является гибберелловая кислота (GA3).
Физиологический эффект GA3 на рост растений хорошо известен,
поскольку он увеличивает концентрацию растворимых углеводов во время
прорастания семян и роста растений.
2. In Vitro Propagation of Endangered Orchid, Vanda pumila Hook.f.
through Protocorms Culture. Sabitri Maharjan, Shreeti Pradhan, Bir
Bahadur Thapa, Bijaya Pant*. American Journal of Plant Sciences, 2019,
10, 1220-1232. Plant Biotechnology Laboratory, Central Department of
Botany, Tribhuvan University, Kirtipur, Nepal.

Today I want to tell you some words about the article I`ve read recently. It
describes the growing of endangered orchid, Vanda pumila Hook.f. In 2019, a
group of botanical scientists, Sabitri Maharjan, Shreeti Pradhan, Bir Bahadur
Thapa and Bijaya Pant published the results of research in American Journal of
Plant Sciences. The main aim was to standardize the protocol for mass
propagation and ex-situ conservation of this specie.
[ Immature seeds of Vanda pumila, collected in the botanical garden of
Kathmandu were taken as a material for the research. After sterilization and
immersion in ethanol, the capsules were cut and the seeds were transferred to a
basal medium with an agar gel. For germination, an MS media of various
strengths was used. Eight-month-old protocorm were cultured with the addition
of the hormones BAP, kinetin, and GA3. A slightly acidic environment in cold
light, observing the 16/8 light/dark mode were used. After 12 weeks of in vitro
growth, the protocorm sprouts began to take root. During the four weeks of
cultivation, the proto-corm in all the tested conditions multiplied and showed the
rudiments of the leaves. The most effective breeding medium was MS of the
greatest strength, giving the longest sprouts. Among the conditions tested with
auxin, it was found that this medium provides the maximum number and length
of Vanda pumila roots. Other studies have come to the same or similar result. ]
The research concluded that among the different tested conditions for shoot
development, the 1/2 MS medium fortified with 1.0 mg/L Kn plus 10% CW and
2.0 mg/L BAP plus 10% CW was found to be the more effective for the
proliferation of large number of shoots (9.50 ± 0.29 shoots per culture) and shoot
length (0.78 ± 0.07 cm per culture) respectively from the in vitro culture
protocorms of Vanda Pumila. Hence the present result could be useful for the
establishment of the standard protocol for the mass propagation and ex-situ
conservation of this endangered orchid species.
 For me, this article, as well as the given research data, were not sufficiently
informative. The in vitro propagation of orchids has always been envisaged as
problematic since their nutrient requirements have not been fully understood. We
still have a need in a specific research for each type of orchid to find the most
effective and ergonomic conditions for their growing and cultivation. As for me,
I`m making the similar research for my diploma thesis and haven`t found
anything, that might be useful in this article.

Материалом для исследования послужили незрелые семена Vanda pumila,

собранные в ботаническом саду Катманду. После стерилизации и
погружения в этанол, капсулы разрезали и перенесли семена в питательную
среду с агаровым гелем. Для проращивания использовали среду MS
различной силы. Восьмимесячные протокормы культивировали с
добавлением гормонов BAP, кинетина и GA3. Использовали слабокислую
среду при холодном освещении, соблюдая режим 16/8 свет/темнота. Спустя
12 недель роста in vitro ростки протокорма начали укореняться. В течение
четырех недель культивирования протокормы во всех тестируемых
условиях размножились и показали зачатки листьев. Наиболее
эффективной для размножения оказалась среда MS наибольшей силы, дав
наиболее длинные ростки. Среди испытанных условий с ауксином было
обнаружено, что эта среда обеспечивает максимальное количество и длину
корней Vanda pumila. Другие исследования пришли к такому же или
похожему результату.
1.In vitro germination, protocorm formation, and plantlet development of
Orchis coriophora (Orchidaceae), a naturally growing orchid species in
Turkey

Today I want to tell you some words about the article I`ve read recently. It
describes the growing of Orchis coriophora, which is naturally growed in Turkey,
its germination in vitro, protocorm formation and plantlet development. In 2013,
a group of botanical scientists, Ersan BEKTAŞ*, Mustafa CÜCE and Atalay
SÖKMEN published the results of research in Turkish Journal of Botany. The
main aim was to find a solution to the problem of orchid growth in an artificial
environment.
[ In recent years, the problem of conserving orchids in nature is quite actual.
The number of orchid species is rapidly declining, the low rate of distribution and
careless harvesting have led to genetic and ecological erosion. Biologists are
forced to look for new methods of cultivation, since the existing ones are suitable
only for a limited number of species. The wild-growing Orchid coriophora was
chosen as the material for the research.
Viable mature seeds, which were prepaired in advance, were placed in
different basal environments and supplemented with 2% sucrose. To achieve a
high rate of germination and formation of protocorm, one of the media was
modified with PGRs and activated carbon. For the purity of the research, the
germination process was divided into 6 stages: from the pregermination stage,
when the embryo swells to feel the seed coat, till the “shoot” stage when the shoot
differs from the protocorm.
After 8 weeks, protocorm was transferred to a fresh media with repeated
addition of activated carbon and different concentrations of cytokinins. Next, the
protocorm was incubated in the growth chamber under a white fluorescent lamp.
10 replicas of 100 mature seeds were used in the experiment. The effect of
cytokinins on the formation of roots and shoots was recorded. Germination data
was analyzed using the Duncan`s test.
Mature seeds were tested for tetrazolium. The viability was approximately
54%. The most effective was the Orchimax basal medium with a germination rate
of approximately 27%. Less effective was LM media (12%). It is assumed that it
was the source of organic nitrogen contained in Orchidmax that contributed to
the germination of the seeds. It is assumed that the addition of activated carbon
and the observance of the photoperiod 16/8 light/dark is an appropriate condition.
No medium was found to be sufficient without the addition of PGR. Cyclotines
did not have a positive effect on germination. The embryos can reproduce in vitro
without mycorrhiza. ]
In conclusion it is said that an appropriate basal medium and an appropriate
PGR will increase the germination rate of Orchid coriophora protocorm in vitro.
They suggest that this research can serve as a starting point for a mass
reproduction and producing of synthetic seeds.
For me, this article, as well as the given research data, were not sufficiently
informative. The in vitro propagation of orchids has always been envisaged as
problematic since their nutrient requirements have not been fully understood. We
still have a need in a specific research for each type of orchid to find the most
effective and ergonomic conditions for their growing and cultivation. As for me,
I`m making the similar research for my diploma thesis and haven`t found
anything, that might be useful in this article.

За последние годы проблема сохранения орхидей в природе стоит

достаточно остро. Количество видов орхидей быстро сокращается, низкая
скорость распространения и и небрежный сбор привел к генетической и
экологической эрозии. Биологи вынуждены искать новые методы
культивации, поскольку уж имеющиеся подходят лишь ограниченному
количеству видов. В качестве материала для исследования была выбрана
дикорастущая Orchid coriophora.
Предварительно подготовленные жизнеспособные зрелые семена
поместили в разные базальные среды, дополненные 2% сахарозой. Для
достижения высокой скорости прорастания и образования протокорм, одну
из сред модифицировали PGR и активированным углем. Для чистоты
исследования весь процесс прорастания разделили на 6 этапов: от
начального, когда рост эмбриона еще не происходит, до этапа, когда побег
отличается от протокорма.
Спустя 8 недель протокормы перенесли в свежие среды с повторный
добавлением активированного угля и разной концентрации цитокининов.
Далее протокормы инкубировали в камере под белой флуоресцентной
лампой.
В эксперименте использовали 10 реплик по 100 зрелых семян.
Регистрировали влияние цитокининов на формирование корней и побегов.
Данные о прорастании анализировали с использованием теста Дункана.
Зрелые семена были подвергнуты испытанию на тетразолий.
Жизнеспособность составила примерно 54%. Наиболее эффективной
оказалась базальная среда Orchimax со всхожестью примерно 27%.
Наименее эффективно показала себя среда LM (12%). Предполагается, что
именно источник органического азота, содержащийся в Orchidmax
способствовал прорастанию семян. Предполагается, что добавление
активированного угля и соблюдение фотопериода 16/8 свет/темнота
является подходящим условием. Ни одна среда не оказалось достаточной
без добавления PGR. Цикотины не оказали положительного влияние на
прорастание. Зародыши могут размножаться in vitro без микоризы.
В заключении статьи ученые приходят к выводу, что правильно
подобранные среда и PGR увеличат скорость прорастания семян Orchid
coriophora in vitro. Они предполагают, что приведенное исследование
может служить отправной точкой для массового размножения и
производства синтетических семян.